Proc. Nail. Acad. Sci. USA Vol. 89, pp. 10395-10399, November 1992
Biochemistry
Zinc rapidly induces a metal response element-binding factor (DNA-protein interaction/heavy metal ions/moblty-shift assay/detection of DNA-binding proteins/UV crosslinking)
MARTA CZUPRYN*, WILLIS E. BROWN, AND BERT L. VALLEEt Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, 250 Longwood Avenue, Boston, MA 02115
Contributed by Bert L. Vallee, July 29, 1992
Metal activation of metallothionein gene ABSTRACT transcription is mediated by specific promoter sequences, termed metal regulatory elements (MREs). Nuclear extracts prepared from various human cell lines were assayed for their capacity to bind to a synthetic human MREa (hMREa) oligomer. Electrophoretic mobility-shift assays with extracts from control cells detected a single hMREa-containing complex. Addition to the growth medium of zinc, cadmium, or coppermetals known to induce MT biosynthesis in vivo-resulted in the rapid but reversible appearance of a second distinct hMREa-protein complex in all cell lines studied. This result was not seen when the metals were added directly to the extracts from control cells. DNA-binding protein blotting, UV crosslinking, and electroelution experiments were used to characterize the two hMREa-binding factors, termed BF1 and BF2. MRE-BF1 has an apparent molecular mass of '46 kDa and binds to the hMREa in control cells, whereas MRE-BF2 consists of two molecules of -28 kDa and binds to the hMREa in metal-treated cells. EDTA and o-phenanroline inhibited binding of both factors to hMREa in a dose-dependent manner, indicating that a metal atom or atoms are essential for interaction of the factors with DNA.
Zinc has long been known to be required for development, differentiation, and gene expression (1, 2). It is an integral component of RNA polymerase (2) and has recently been shown to be an essential component of numerous transcription factors (3). In addition, zinc directly affects the expression of metallothioneins (MTs) (4), small, cysteine-rich, zinc (and cadmium)-binding proteins thought to regulate metal homeostasis (5, 6). Zinc (and other heavy metal) induction of MT occurs at the transcriptional level (4, 7) and is mediated by metal regulatory elements (MREs) located upstream from the transcription start site of the mammalian MT-encoding genes (8-10). MREs are present in multiple (five or more) nonidentical copies per gene, and all known elements contain the 7-nucleotide consensus core sequence TGCRCNC (11). MREs are believed to mediate metal-induced transcription of MT genes via interaction with specific, trans-acting regulatory factors. Metal-dependent binding of regulatory factors to MREs has been observed by genomic footprinting in vivo (12) and by electrophoretic mobility-shift assays (12-15), UV crosslinking (15-17), DNA-binding protein blotting (17, 18), and DNase 1 (16) or exonuclease III (12, 17-19) footprinting in vitro. All of these studies were done under static conditions-i.e., using cells either grown under normal conditions or exposed to a single concentration of heavy metals, making any correlation between MRE binding and transcription activation difficult. The present study was undertaken to determine what, if any, changes occur in factors that bind to the human MREa (hMREa) when heavy metals induce MT biosynthesis. The results demonstrate the reversible appearance of a distinct MRE-binding factor (BF) in cells treated
with zinc, cadmium, or copper, which differs from the one present in untreated cells. This phenomenon was seen in all cell lines studied and could not be induced by the addition of metals in vitro.
MATERIALS AND METHODS Cells and Nuclear Extracts. Human HepG2, hepatocellular carcinoma; A549, lung carcinoma; HL-60, promyelocytic leukemia; and K-562, chronic myelogenous leukemia cell lines purchased from American Type Culture Collection, were maintained in Dulbecco's modified Eagle's medium (Whittaker Bioproducts)/10%o fetal calf serum (HyClone)/2 mM L-glutamine/gentamicin at 25 ug/ml. For metal treatment, sterile solutions of ZnSO4, CdSO4, or CuSO4 at the concentrations indicated were added to the medium for 0.5-24 hr before cell harvesting. Nuclear extracts from tissue culture cells were prepared as described (20). Protein concentration was determined by the BCA protein assay (Pierce). DNA Probes and Competitor Fragments. Oligonucleotides (hMREa, 5'-CGTCZiCfLCCCGGCCCCGTC-3' and mouse MREd, 5'-CGATCTCTGCACTCCGCCCGA-3') and their complements were synthesized on a Biotix (Danbury, CT) DNA 102 synthesizer and hybridized as described (21). For electrophoretic mobility-shift assays, the hMREa oligomer was end-labeled by using T4 polynucleotide kinase and [y-32P]ATP (NEN). For UV-crosslinking experiments, hMREa concatemers were prepared and nick-translated by using [a-32P]dCTP (NEN) as described (21). Electrophoretic Mobility-Shift Assay. Gel-shift assays were done as a modification of earlier methods (22, 23). Binding reaction mixtures (20 1.l) contained 12 mM Hepes, pH 7.9/5 mM NaCl/50 mM KCl/5 mM MgCl2/0.6 mM dithiothreitol (binding buffer)/12% (vol/vol) glycerol/radiolabeled hMRE probe (40-160 fmol, 104-4 x 104 cpm)/0.4 ,ug of doublestranded poly(dI-dC) (Pharmacia)/5-10 ,ug of nuclear extract total protein. Reaction mixtures were incubated at 250C for 20 min and then resolved on 7% polyacrylamide gels (acrylamide/bisacrylamide, 29:1) in 44.5 mM Tris/44.5 mM boric acid. Gels were run for 2.5 hr at 7.5 V/cm, dried, and exposed to Kodak X-Omat XAR-5 film. Blotting of DNA-binding Proteins, UV Crosslinking, and Electroelution. Nuclear extracts were resolved on an SDS/ 10% polyacrylamide gel according to ref. 24 as modified (25). The proteins were transferred to a nitrocellulose membrane (Schleicher & Schuell) in 25 mM Tris/0.2 M glycine, pH 8.6, for 5 hr at 5 V/cm. Membranes were hybridized and washed as in ref. 25 by using a modified hybridization buffer (5% nonfat milk/10 mM Hepes, pH 7.9/100 ,uM ZnSO4/0.6 mM dithiothreitol) and labeled hMRE fragment (10W cpm/ml). UV Abbreviations: OP, o-phenanthroline; MRE, metal regulatory element; MT, metallothionein; BF, binding factor; hMREa, human MREa.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
*Present address: Genetics Institute, 1 Burtt Road, Andover, MA 01810. tTo whom reprint requests should be addressed. 10395
103%
Proc. Natl. Acad Sci. USA 89 (1992)
Biochemistry: Czupryn et al.
crosslinking was done by a modification of Safer et al. (26). Nuclear extracts (=10 Ag of total protein) were mixed with 32p nick-translated hMRE concatemers in the binding buffer (total vol of 20 ull) and 4 jig of poly(dl dC), incubated at 250C for 10 min, exposed to a short-wavelength UV light at 40C for 15 min, and then electrophoresed on an SDS/15% polyacrylamide gel (24). Gels were dried under vacuum and autoradiographed. For electroelution a 100-Al binding reaction was applied in 20-jil aliquots into five adjacent wells of a 5% polyacrylamide gel and electrophoresed, as described for the mobility-shift assay. The radioactive bands were visualized by exposing the wet gel to x-ray film for 3 hr at 40C; hMREa complexes were excised from the gel and electroeluted into 22 mM Tris/22 mM boric acid. Samples were dialyzed against water, lyophilized, then analyzed by SDS/PAGE, and visualized by silver staining (27).
RESULTS Identification of Two Distinc hMREa-BFs in HepG2 Cells. Because the liver is one of the primary sites of MT synthesis in adults (28), initial experiments were done with human hepatoma cell line HepG2. These cells respond to heavy metal ions by increasing synthesis of MT mRNA (29) and, therefore, should contain MT gene-specific transcription factors. A labeled synthetic 20-nucleotide fragment corresponding to the hMRE element proximal to the TATA box, hMREa, was used. This element was chosen because its consensus core sequence TGCGCCC, as well as its position within the MT gene promoter, are conserved among five human MT-I-encoding genes (see ref. 30, figure 8). This sequence is also present in the MREd of the hMT-IIA gene (31). Deletion analysis of the human MT gene promoters have shown that the proximal MREs are functional in vivo (8, 32). Incubation of the hMREa probe with a nuclear extract prepared from HepG2 cells grown in the absence of added zinc generated a distinct complex designated C-1 (Fig. 1A). Another minor complex, with an electrophoretic mobility slower than that of C-1, was present only occasionally and was, therefore, not studied further. When the growth medium was supplemented with zinc (50-500 tuM ZnSO4) for 2 hr, an additional, faster-migrating complex, C-2, was seen (Fig. 1A). This metal-induced complex was readily observed in
C
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Ci-Cl-~~Ci UX!C1-WB 0 C21 i C2 C2t
Free_
probe
0
°
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OO
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cells incubated with 50 ,uM ZnSO4 and was more intense when higher zinc concentrations were used. Concurrent with the increase in complex C-2, the amount of complex C-1 decreased. At 500 ILM ZnSO4, complex C-1 was often not seen at all. The induction of complex C-2 by zinc and the concomitant decrease of complex C-1 occurred within the first 30 min of incubation of HepG2 cells with 500 juM ZnSO4 (Fig. 1B). Densitometric scanning of the autoradiogram indicated that complex C-2 reached a maximum 2 hr after incubating cells with zinc. Upon prolonged incubation, the amount of complex C-2 appeared to decrease (Fig. 1B). This phenomenon was reversible. When HepG2 cells grown in zinc-supplemented medium were transferred to normal medium, the amount of complex C-2 diminished (Fig. 1C). After 12 hr, only complex C-1 could be seen (Fig. 1C). Lung tissue also responds to heavy metals by synthesizing MT (33). Incubation of nuclear extracts prepared from uninduced and zinc-induced A549 lung carcinoma cells with the hMREa probe resulted in the formation of complexes that had the same electrophoretic mobilities as C-1 and C-2 from HepG2 cells (Fig. 2). Induction of complex C-2 by zinc occurred at the same metal concentration and proceeded with comparable kinetics (data not shown). Similar DNA-protein complexes were also seen in nuclear extracts from human leukemic cells K-562 and HL-60, grown with and without added zinc (data not shown). These results indicate that the same (or similar) nuclear factors are involved in the interaction with the hMREa in a variety of human tissues (as confirmed by the DNA-binding protein blotting experiments, see below). Because A549 cells grew most readily under our cell culture conditions, these cells were used as a source of nuclear extracts in most experiments described below. The intensity of both complex C-1 and C-2 decreased upon adding a 50- to 250-fold molar excess of the unlabeled hMREa fragment to the binding reactions (data not shown). In contrast, a mouse MREd oligonucleotide (containing motif TGCACTC, which differs from the hMREa consensus in two positions) had no effect on the formation of the complexes, even with a 250-fold excess of the fragment (data not shown). These data indicate that hMREa complex formation is sequence specific. Induction of Activated Complex Is Mediated by Various Metal Ions and Occurs in Vivo but Does Not Occur in Vitro. The metal-mediated formation of complex C-2 was also seen when cells were exposed to cadmium or copper, metals that induce MT gene transcription in vivo (for review, see ref. 4). Nuclear extracts prepared from A549 cells grown with either Zn, Cd, CuJM °o
o
C1123
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cm0
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++
C2-
Zn, pM
FIG. 1. Zinc induces a hMREa-BF that differs from that in control cells. Nuclear extract protein (9-12 pug) was incubated with 40 fmol of labeled hMREa and analyzed by using mobility-shift assay. The electrophoretic mobility of complex C-1 in the control, the mobility of complex C-2 induced by zinc, and the mobility of the free oligomer probe are indicated. (A) Concentration-dependence. HepG2 cells grown with or without the indicated zinc concentrations for 2 hr. (B) Time course for zinc induction of complex C-2. HepG2 cells were incubated with 500 AtM ZnSO4 for the indicated periods. (C) Disappearance of complex C-2 after removal of zinc from the medium. Cells were incubated in normal medium (lane 1); medium containing 400 1uM ZnSO4 for 2 hr (lane 2); medium containing 400 1AM ZnSO4 for 2 hr and then in normal medium for 12 hr (lane 3).
Free_ probe 1 2 3 4 5 6 FIG. 2. Zinc, cadmium, and copper induction of complex C-2. A549 cells were incubated with or without the indicated concentrations of metals for 2 hr. Nuclear extract protein (4.5 ,.g) was assayed for its capacity to bind hMREa by the mobility-shift assay. Lanes: 1, 3, and 5, extracts prepared from cells incubated without metals; 2, 4, and 6, extracts prepared from cells incubated with 500 A&M ZnSO4, 20 ,AM CdSO4, or 200 ,AM CuCl2, respectively.
Proc. Nati. Acad. Sci. USA 89 (1992)
Biochemistry: Czupryn et al. 20 ,uM CdSO4 or 200 AtM CuCl2 contained a factor that recognized the hMREa oligonucleotide and formed a complex with the same electrophoretic mobility as the zincinduced complex C-2 (Fig. 2). To determine whether complex C-2 can be induced in vitro, a nuclear extract from control A549 cells was incubated for 30 min with 100-500 ,uM ZnSO4 or 5-20 ,uM CdSO4, and hMREa-binding activity was analyzed by the mobility-shift assay. Addition of zinc or cadmium did not result in the appearance of complex C-2, nor did the intensity of complex C-1 change (data not shown). These results suggest that C-2 does not arise from C-1 as a result of structural changes generated in the latter complex by the metal, but rather that the C-1 and C-2 complexes originate by the binding ofhMREa to two different factors. The factor that gives rise to complex C-1 was termed MRE-BF1 and that involved in the formation of complex C-2, MRE-BF2. Characterization of the hMR~a BFs. To characterize the hMREa BFs, DNA-binding protein blotting and UVcrosslinking experiments were done on nuclear extracts from control and zinc-induced cells. For DNA-binding protein blotting, nuclear extracts were prepared from control and metal-induced HepG2, A549, HL-60, and K-562 cells. In the control extracts, the predominant complex was a band of 86 kDa (Fig. 3). Consistent with the disappearance of complex C-1 observed by the mobility-shift assays, extracts from metal-treated cells lacked this band, which, therefore, was most likely MRE-BF1. The only hMREa-binding species in induced extracts contained two closely migrating bands of -28 kDa (Fig. 3), probably MRE-BF2. The size of MRE-BF1 and MRE-BF2 detected by DNA-binding protein blotting was identical in all cell lines studied. Although MRE-BF2 binding activity was predominant in metal-induced cells (Fig. 3), small amounts of MRE-BF2 were detected in the control extracts by this technique, in contrast to the mobility-shift assays (Figs. 1 and 2) or UV crosslinking (Fig. 4A). The reducing/denaturing conditions used in DNA-binding protein blotting could possibly dissociate MRE-BF2 from an inhibitor that prevented hMREa binding. Alternatively, immobilization on the membrane could change the DNA-binding properties of MRE-BF2. Nevertheless, this result suggests that the factor is present in uninduced cells. UV crosslinking of the labeled hMREa probe to components of nuclear extracts revealed a single complex migrating as a species of =90 kDa in the control A549 cells (Fig. 4A). Because the electrophoretic mobility of proteins in SDS gels is only affected slightly by covalent attachment of short DNA fragments (27), the size of this complex agrees well with the
A
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size of MRE-BF1. In the extracts from metal-induced cells, MRE-BF1 was not detected. Instead, two UV-crosslinked complexes of -32 kDa and -29 kDa were present. These complexes likely correspond to the components ofMRE-BF2 identified by DNA-binding protein blotting. The assignment of hMREa-BFs was confirmed by direct electrophoretic analysis of protein components of complexes C-1 and C-2, electroeluted from a polyacrylamide gel. Fig. 4B shows that complex C-1 contained a single protein of -86 kDa (MRE-BF1), whereas complex C-2 contained a protein doublet migrating as w28-kDa species (MRE-BF2). The relative affinity of MRE-BF1 and MRE-BF2 for the hMREa oligomer was assessed by performing the mobilityshift assay with a large excess of poly(dI-dC) (15). To achieve a 50% decrease in C-1 and C-2 complexes 1.5 ,ug and 8 j&g of poly(dIdC) were required, respectively (data not shown). These results suggest that MRE-BF2 binds to hMREa with a higher affinity than does MRE-BF1. Binding of both MRE-BF1 and MRE-BF2 to the hMREa probe appeared metal-dependent. Although addition of exogenous metals to the binding reaction had no effect on hMREa binding, preincubation of the extracts with EDTA or o-phenanthroline (OP) inhibited formation ofboth complexes C-1 and C-2 in a concentration-dependent fashion (Fig. 5). The extent ofinhibition was similar for both: '-80% of binding was abolished by a 20-min incubation of the extracts with 10 mM EDTA or 10 mM OP. This result suggests that a metal ion is essential for DNA binding of both MRE-BF1 and -BF2.
DISCUSSION Two distinct nuclear factors from human cells, MRE-BF1 and MRE-BF2, interact with the hMREa of the MT-I genes.
A EDTA, mM OP, mM
B EDTA, mM OP, mM
0 0.2 2 10 0.22 10
00.22 10 0.22 10
C1- Wt
%d C2-
*,i" a
I
30-
FIG. 4. Detection of nuclear factors that bind to hMREa. A549 cells were incubated with or without 500 ,uM ZnSO4 for 2 hr. (A) UV crosslinking. (B) Electroelution of complexes. Molecular mass markers are indicated. Bracket indicates staining artifacts due to 2-mercaptoethanol (34).
T
946743-
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-Zn + Zn
00 ~~0
Biochemistry
Zinc rapidly induces a metal response element-binding factor (DNA-protein interaction/heavy metal ions/moblty-shift assay/detection of DNA-binding proteins/UV crosslinking)
MARTA CZUPRYN*, WILLIS E. BROWN, AND BERT L. VALLEEt Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, 250 Longwood Avenue, Boston, MA 02115
Contributed by Bert L. Vallee, July 29, 1992
Metal activation of metallothionein gene ABSTRACT transcription is mediated by specific promoter sequences, termed metal regulatory elements (MREs). Nuclear extracts prepared from various human cell lines were assayed for their capacity to bind to a synthetic human MREa (hMREa) oligomer. Electrophoretic mobility-shift assays with extracts from control cells detected a single hMREa-containing complex. Addition to the growth medium of zinc, cadmium, or coppermetals known to induce MT biosynthesis in vivo-resulted in the rapid but reversible appearance of a second distinct hMREa-protein complex in all cell lines studied. This result was not seen when the metals were added directly to the extracts from control cells. DNA-binding protein blotting, UV crosslinking, and electroelution experiments were used to characterize the two hMREa-binding factors, termed BF1 and BF2. MRE-BF1 has an apparent molecular mass of '46 kDa and binds to the hMREa in control cells, whereas MRE-BF2 consists of two molecules of -28 kDa and binds to the hMREa in metal-treated cells. EDTA and o-phenanroline inhibited binding of both factors to hMREa in a dose-dependent manner, indicating that a metal atom or atoms are essential for interaction of the factors with DNA.
Zinc has long been known to be required for development, differentiation, and gene expression (1, 2). It is an integral component of RNA polymerase (2) and has recently been shown to be an essential component of numerous transcription factors (3). In addition, zinc directly affects the expression of metallothioneins (MTs) (4), small, cysteine-rich, zinc (and cadmium)-binding proteins thought to regulate metal homeostasis (5, 6). Zinc (and other heavy metal) induction of MT occurs at the transcriptional level (4, 7) and is mediated by metal regulatory elements (MREs) located upstream from the transcription start site of the mammalian MT-encoding genes (8-10). MREs are present in multiple (five or more) nonidentical copies per gene, and all known elements contain the 7-nucleotide consensus core sequence TGCRCNC (11). MREs are believed to mediate metal-induced transcription of MT genes via interaction with specific, trans-acting regulatory factors. Metal-dependent binding of regulatory factors to MREs has been observed by genomic footprinting in vivo (12) and by electrophoretic mobility-shift assays (12-15), UV crosslinking (15-17), DNA-binding protein blotting (17, 18), and DNase 1 (16) or exonuclease III (12, 17-19) footprinting in vitro. All of these studies were done under static conditions-i.e., using cells either grown under normal conditions or exposed to a single concentration of heavy metals, making any correlation between MRE binding and transcription activation difficult. The present study was undertaken to determine what, if any, changes occur in factors that bind to the human MREa (hMREa) when heavy metals induce MT biosynthesis. The results demonstrate the reversible appearance of a distinct MRE-binding factor (BF) in cells treated
with zinc, cadmium, or copper, which differs from the one present in untreated cells. This phenomenon was seen in all cell lines studied and could not be induced by the addition of metals in vitro.
MATERIALS AND METHODS Cells and Nuclear Extracts. Human HepG2, hepatocellular carcinoma; A549, lung carcinoma; HL-60, promyelocytic leukemia; and K-562, chronic myelogenous leukemia cell lines purchased from American Type Culture Collection, were maintained in Dulbecco's modified Eagle's medium (Whittaker Bioproducts)/10%o fetal calf serum (HyClone)/2 mM L-glutamine/gentamicin at 25 ug/ml. For metal treatment, sterile solutions of ZnSO4, CdSO4, or CuSO4 at the concentrations indicated were added to the medium for 0.5-24 hr before cell harvesting. Nuclear extracts from tissue culture cells were prepared as described (20). Protein concentration was determined by the BCA protein assay (Pierce). DNA Probes and Competitor Fragments. Oligonucleotides (hMREa, 5'-CGTCZiCfLCCCGGCCCCGTC-3' and mouse MREd, 5'-CGATCTCTGCACTCCGCCCGA-3') and their complements were synthesized on a Biotix (Danbury, CT) DNA 102 synthesizer and hybridized as described (21). For electrophoretic mobility-shift assays, the hMREa oligomer was end-labeled by using T4 polynucleotide kinase and [y-32P]ATP (NEN). For UV-crosslinking experiments, hMREa concatemers were prepared and nick-translated by using [a-32P]dCTP (NEN) as described (21). Electrophoretic Mobility-Shift Assay. Gel-shift assays were done as a modification of earlier methods (22, 23). Binding reaction mixtures (20 1.l) contained 12 mM Hepes, pH 7.9/5 mM NaCl/50 mM KCl/5 mM MgCl2/0.6 mM dithiothreitol (binding buffer)/12% (vol/vol) glycerol/radiolabeled hMRE probe (40-160 fmol, 104-4 x 104 cpm)/0.4 ,ug of doublestranded poly(dI-dC) (Pharmacia)/5-10 ,ug of nuclear extract total protein. Reaction mixtures were incubated at 250C for 20 min and then resolved on 7% polyacrylamide gels (acrylamide/bisacrylamide, 29:1) in 44.5 mM Tris/44.5 mM boric acid. Gels were run for 2.5 hr at 7.5 V/cm, dried, and exposed to Kodak X-Omat XAR-5 film. Blotting of DNA-binding Proteins, UV Crosslinking, and Electroelution. Nuclear extracts were resolved on an SDS/ 10% polyacrylamide gel according to ref. 24 as modified (25). The proteins were transferred to a nitrocellulose membrane (Schleicher & Schuell) in 25 mM Tris/0.2 M glycine, pH 8.6, for 5 hr at 5 V/cm. Membranes were hybridized and washed as in ref. 25 by using a modified hybridization buffer (5% nonfat milk/10 mM Hepes, pH 7.9/100 ,uM ZnSO4/0.6 mM dithiothreitol) and labeled hMRE fragment (10W cpm/ml). UV Abbreviations: OP, o-phenanthroline; MRE, metal regulatory element; MT, metallothionein; BF, binding factor; hMREa, human MREa.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
*Present address: Genetics Institute, 1 Burtt Road, Andover, MA 01810. tTo whom reprint requests should be addressed. 10395
103%
Proc. Natl. Acad Sci. USA 89 (1992)
Biochemistry: Czupryn et al.
crosslinking was done by a modification of Safer et al. (26). Nuclear extracts (=10 Ag of total protein) were mixed with 32p nick-translated hMRE concatemers in the binding buffer (total vol of 20 ull) and 4 jig of poly(dl dC), incubated at 250C for 10 min, exposed to a short-wavelength UV light at 40C for 15 min, and then electrophoresed on an SDS/15% polyacrylamide gel (24). Gels were dried under vacuum and autoradiographed. For electroelution a 100-Al binding reaction was applied in 20-jil aliquots into five adjacent wells of a 5% polyacrylamide gel and electrophoresed, as described for the mobility-shift assay. The radioactive bands were visualized by exposing the wet gel to x-ray film for 3 hr at 40C; hMREa complexes were excised from the gel and electroeluted into 22 mM Tris/22 mM boric acid. Samples were dialyzed against water, lyophilized, then analyzed by SDS/PAGE, and visualized by silver staining (27).
RESULTS Identification of Two Distinc hMREa-BFs in HepG2 Cells. Because the liver is one of the primary sites of MT synthesis in adults (28), initial experiments were done with human hepatoma cell line HepG2. These cells respond to heavy metal ions by increasing synthesis of MT mRNA (29) and, therefore, should contain MT gene-specific transcription factors. A labeled synthetic 20-nucleotide fragment corresponding to the hMRE element proximal to the TATA box, hMREa, was used. This element was chosen because its consensus core sequence TGCGCCC, as well as its position within the MT gene promoter, are conserved among five human MT-I-encoding genes (see ref. 30, figure 8). This sequence is also present in the MREd of the hMT-IIA gene (31). Deletion analysis of the human MT gene promoters have shown that the proximal MREs are functional in vivo (8, 32). Incubation of the hMREa probe with a nuclear extract prepared from HepG2 cells grown in the absence of added zinc generated a distinct complex designated C-1 (Fig. 1A). Another minor complex, with an electrophoretic mobility slower than that of C-1, was present only occasionally and was, therefore, not studied further. When the growth medium was supplemented with zinc (50-500 tuM ZnSO4) for 2 hr, an additional, faster-migrating complex, C-2, was seen (Fig. 1A). This metal-induced complex was readily observed in
C
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Free_
probe
0
°
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cells incubated with 50 ,uM ZnSO4 and was more intense when higher zinc concentrations were used. Concurrent with the increase in complex C-2, the amount of complex C-1 decreased. At 500 ILM ZnSO4, complex C-1 was often not seen at all. The induction of complex C-2 by zinc and the concomitant decrease of complex C-1 occurred within the first 30 min of incubation of HepG2 cells with 500 juM ZnSO4 (Fig. 1B). Densitometric scanning of the autoradiogram indicated that complex C-2 reached a maximum 2 hr after incubating cells with zinc. Upon prolonged incubation, the amount of complex C-2 appeared to decrease (Fig. 1B). This phenomenon was reversible. When HepG2 cells grown in zinc-supplemented medium were transferred to normal medium, the amount of complex C-2 diminished (Fig. 1C). After 12 hr, only complex C-1 could be seen (Fig. 1C). Lung tissue also responds to heavy metals by synthesizing MT (33). Incubation of nuclear extracts prepared from uninduced and zinc-induced A549 lung carcinoma cells with the hMREa probe resulted in the formation of complexes that had the same electrophoretic mobilities as C-1 and C-2 from HepG2 cells (Fig. 2). Induction of complex C-2 by zinc occurred at the same metal concentration and proceeded with comparable kinetics (data not shown). Similar DNA-protein complexes were also seen in nuclear extracts from human leukemic cells K-562 and HL-60, grown with and without added zinc (data not shown). These results indicate that the same (or similar) nuclear factors are involved in the interaction with the hMREa in a variety of human tissues (as confirmed by the DNA-binding protein blotting experiments, see below). Because A549 cells grew most readily under our cell culture conditions, these cells were used as a source of nuclear extracts in most experiments described below. The intensity of both complex C-1 and C-2 decreased upon adding a 50- to 250-fold molar excess of the unlabeled hMREa fragment to the binding reactions (data not shown). In contrast, a mouse MREd oligonucleotide (containing motif TGCACTC, which differs from the hMREa consensus in two positions) had no effect on the formation of the complexes, even with a 250-fold excess of the fragment (data not shown). These data indicate that hMREa complex formation is sequence specific. Induction of Activated Complex Is Mediated by Various Metal Ions and Occurs in Vivo but Does Not Occur in Vitro. The metal-mediated formation of complex C-2 was also seen when cells were exposed to cadmium or copper, metals that induce MT gene transcription in vivo (for review, see ref. 4). Nuclear extracts prepared from A549 cells grown with either Zn, Cd, CuJM °o
o
C1123
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cm0
0 CM
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Zn, pM
FIG. 1. Zinc induces a hMREa-BF that differs from that in control cells. Nuclear extract protein (9-12 pug) was incubated with 40 fmol of labeled hMREa and analyzed by using mobility-shift assay. The electrophoretic mobility of complex C-1 in the control, the mobility of complex C-2 induced by zinc, and the mobility of the free oligomer probe are indicated. (A) Concentration-dependence. HepG2 cells grown with or without the indicated zinc concentrations for 2 hr. (B) Time course for zinc induction of complex C-2. HepG2 cells were incubated with 500 AtM ZnSO4 for the indicated periods. (C) Disappearance of complex C-2 after removal of zinc from the medium. Cells were incubated in normal medium (lane 1); medium containing 400 1uM ZnSO4 for 2 hr (lane 2); medium containing 400 1AM ZnSO4 for 2 hr and then in normal medium for 12 hr (lane 3).
Free_ probe 1 2 3 4 5 6 FIG. 2. Zinc, cadmium, and copper induction of complex C-2. A549 cells were incubated with or without the indicated concentrations of metals for 2 hr. Nuclear extract protein (4.5 ,.g) was assayed for its capacity to bind hMREa by the mobility-shift assay. Lanes: 1, 3, and 5, extracts prepared from cells incubated without metals; 2, 4, and 6, extracts prepared from cells incubated with 500 A&M ZnSO4, 20 ,AM CdSO4, or 200 ,AM CuCl2, respectively.
Proc. Nati. Acad. Sci. USA 89 (1992)
Biochemistry: Czupryn et al. 20 ,uM CdSO4 or 200 AtM CuCl2 contained a factor that recognized the hMREa oligonucleotide and formed a complex with the same electrophoretic mobility as the zincinduced complex C-2 (Fig. 2). To determine whether complex C-2 can be induced in vitro, a nuclear extract from control A549 cells was incubated for 30 min with 100-500 ,uM ZnSO4 or 5-20 ,uM CdSO4, and hMREa-binding activity was analyzed by the mobility-shift assay. Addition of zinc or cadmium did not result in the appearance of complex C-2, nor did the intensity of complex C-1 change (data not shown). These results suggest that C-2 does not arise from C-1 as a result of structural changes generated in the latter complex by the metal, but rather that the C-1 and C-2 complexes originate by the binding ofhMREa to two different factors. The factor that gives rise to complex C-1 was termed MRE-BF1 and that involved in the formation of complex C-2, MRE-BF2. Characterization of the hMR~a BFs. To characterize the hMREa BFs, DNA-binding protein blotting and UVcrosslinking experiments were done on nuclear extracts from control and zinc-induced cells. For DNA-binding protein blotting, nuclear extracts were prepared from control and metal-induced HepG2, A549, HL-60, and K-562 cells. In the control extracts, the predominant complex was a band of 86 kDa (Fig. 3). Consistent with the disappearance of complex C-1 observed by the mobility-shift assays, extracts from metal-treated cells lacked this band, which, therefore, was most likely MRE-BF1. The only hMREa-binding species in induced extracts contained two closely migrating bands of -28 kDa (Fig. 3), probably MRE-BF2. The size of MRE-BF1 and MRE-BF2 detected by DNA-binding protein blotting was identical in all cell lines studied. Although MRE-BF2 binding activity was predominant in metal-induced cells (Fig. 3), small amounts of MRE-BF2 were detected in the control extracts by this technique, in contrast to the mobility-shift assays (Figs. 1 and 2) or UV crosslinking (Fig. 4A). The reducing/denaturing conditions used in DNA-binding protein blotting could possibly dissociate MRE-BF2 from an inhibitor that prevented hMREa binding. Alternatively, immobilization on the membrane could change the DNA-binding properties of MRE-BF2. Nevertheless, this result suggests that the factor is present in uninduced cells. UV crosslinking of the labeled hMREa probe to components of nuclear extracts revealed a single complex migrating as a species of =90 kDa in the control A549 cells (Fig. 4A). Because the electrophoretic mobility of proteins in SDS gels is only affected slightly by covalent attachment of short DNA fragments (27), the size of this complex agrees well with the
A
0
kDaN °
C
B 0
0~ °
~
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D
kDa 94 67 43 -
9
0 -E
-
20 14-
C-1 C-2 kDa 94 67 43 -
20 h
A
14 -
size of MRE-BF1. In the extracts from metal-induced cells, MRE-BF1 was not detected. Instead, two UV-crosslinked complexes of -32 kDa and -29 kDa were present. These complexes likely correspond to the components ofMRE-BF2 identified by DNA-binding protein blotting. The assignment of hMREa-BFs was confirmed by direct electrophoretic analysis of protein components of complexes C-1 and C-2, electroeluted from a polyacrylamide gel. Fig. 4B shows that complex C-1 contained a single protein of -86 kDa (MRE-BF1), whereas complex C-2 contained a protein doublet migrating as w28-kDa species (MRE-BF2). The relative affinity of MRE-BF1 and MRE-BF2 for the hMREa oligomer was assessed by performing the mobilityshift assay with a large excess of poly(dI-dC) (15). To achieve a 50% decrease in C-1 and C-2 complexes 1.5 ,ug and 8 j&g of poly(dIdC) were required, respectively (data not shown). These results suggest that MRE-BF2 binds to hMREa with a higher affinity than does MRE-BF1. Binding of both MRE-BF1 and MRE-BF2 to the hMREa probe appeared metal-dependent. Although addition of exogenous metals to the binding reaction had no effect on hMREa binding, preincubation of the extracts with EDTA or o-phenanthroline (OP) inhibited formation ofboth complexes C-1 and C-2 in a concentration-dependent fashion (Fig. 5). The extent ofinhibition was similar for both: '-80% of binding was abolished by a 20-min incubation of the extracts with 10 mM EDTA or 10 mM OP. This result suggests that a metal ion is essential for DNA binding of both MRE-BF1 and -BF2.
DISCUSSION Two distinct nuclear factors from human cells, MRE-BF1 and MRE-BF2, interact with the hMREa of the MT-I genes.
A EDTA, mM OP, mM
B EDTA, mM OP, mM
0 0.2 2 10 0.22 10
00.22 10 0.22 10
C1- Wt
%d C2-
*,i" a
I
30-
FIG. 4. Detection of nuclear factors that bind to hMREa. A549 cells were incubated with or without 500 ,uM ZnSO4 for 2 hr. (A) UV crosslinking. (B) Electroelution of complexes. Molecular mass markers are indicated. Bracket indicates staining artifacts due to 2-mercaptoethanol (34).
T
946743-
30-
-Zn + Zn
00 ~~0
