nuclear factor-1 and metal transcription factor-1 synergistically

Nuclear Factor-1 and Metal Transcription Factor-1Synergistically Activate the Mouse Metallothionein-1Gene in Response to Metal Ions*

Received for publication, January 24, 2008 Published, JBC Papers in Press, January 29, 2008, DOI 10.1074/jbc.M800640200

Olivier LaRochelle, Simon Labbe1, Jean-Francois Harrisson, Carl Simard, Veronique Tremblay, Genevieve St-Gelais,Manjapra V. Govindan, and Carl Seguin2

From the Centre de recherche en cancerologie de l’Universite Laval, CHUQ, Hotel-Dieu de Quebec, Quebec G1R 2J6 and theDepartement d’anatomie et de physiologie, Faculte de medecine, Universite Laval, Quebec G1K 7P4, Canada

Metal activation of metallothionein (MT) gene transcriptionis dependent on the presence of metal regulatory elements(MREs), which are present in five non-identical copies (MREathroughMREe) in the promoter of themouseMT-1 gene and onthe capacity of metal transcription factor-1 (MTF-1) to bind totheMREs in the presence of zinc.Wedetected a protein, distinctfrom MTF-1, specifically binding to the MREc region. DNAbinding competition experiments using synthetic oligonucleo-tides and specific anti-NF1 antibodies showed that this proteinbinds to an NF1 site overlapping theMREc element as well as toa second site upstream of the Sp1a site and corresponds to NF1or a related protein. Transfection experiments showed that lossof the two NF1 sites decreased metal-induced MT promoteractivity by 55–70% in transiently transfected cells and almostcompletely abrogated metal and tert-butylhydroquinone(tBHQ) induction in stably transfected cells. Similarly, expres-sion of an inactive NF1 protein strongly inhibited MT-1 pro-moter activity. Using synthetic promoters containing NF1 andMRE sites fused to a minimal MT promoter, we showed thatthese NF1 sites did not confer metal induction but enhancedmetal-induced promoter activity. Chromatin immunoprecipi-tation assays confirmed that NF1 binds to themouseMT-1 pro-moter in vivo and showed that NF1 binding is zinc-inducible. Inaddition, zinc-induced NF1 DNA binding was MTF-1-depend-ent. Taken together, these studies show that NF1 acts synergis-tically with MTF-1 to activate the mouse MT-1 promoter inresponse tometal ions and tert-butylhydroquinone and contrib-utes to maximal activation of the gene.

Metallothioneins (MTs)3 are small metal-binding stress pro-teins grouped into four classes, MT-1 through MT-4 (1, 2).

MTs have been identified in a wide range of species and arepresent in various tissues and cell types from yeast to human. Inmice, MT-1 and MT-2 are ubiquitous and coordinatelyexpressed in all tissues, whereas MT-3 is mainly expressed inthe brain (3) and in the organs of the reproductive system (4),andMT-4 is restricted to stratified squamous epithelia (5).MTshave no enzymatic function but appear to play important rolesin metal ion homeostasis, as an active donor of zinc to othersites within the cell, in detoxification of toxic metals, and inprotection against oxidative damage, ionizing radiation, andxenobiotics (1, 2).MT genes are inducible at the transcription level by hor-

mones, cytokines, and a variety of stress conditions that includeexposure to transition metal ions, UV irradiation, hypoxia, andreactive oxygen species (2). Metals are the most general andpotent of these inducers. Metal activation of MT gene tran-scription depends on the presence of regulatory DNAsequences termed metal regulatory elements (MREs) andinvolves metal-responsive transcription factor-1 (MTF-1)interacting with the MREs in a zinc-dependent manner (6, 7).MTF-1 is also involved in the response to hypoxia (8), reactiveoxygen species (9), and amino acid starvation (10). The highlyconserved core sequence 5�-TGCRCNC-3� (R, purine; N, anynucleotide) is necessary and sufficient for induction by metals(11–13). MREs are present in five non-identical copies (MREathrough MREe) in the 5� flanking region of the mouse MT-1gene (Fig. 1), and different MREs have different transcriptionalefficiencies. MREd is the strongest, MREa and MREc are50–80% weaker, MREb is very weak, and MREe is apparentlynon-functional (14). In addition to MTF-1, several other pro-teins interactwith themouseMT-1promoter, includingUSF-1,USF-2 (15–19), Sp1 (9, 19–21), c-Fos (19), and c-Jun (9). How-ever, the mechanism by which these factors contribute to MTgene expression in not known.MTF-1 gene knock-out showed that MTF-1 is essential for

basal and metal-induced MT gene transcription (22). Notably,no MRE-binding protein could be detected in MTF-1 nullmutant cells. This led to the hypothesis that MTF-1 is the onlyfactor that binds MREs and the only transcription factor thatmediates responsiveness to different metals. We previouslyidentified and purified a mouse nuclear protein, termed metal

* This work was supported by a grant from the Conseil de recherches ensciences naturelles et en genie du Canada (to C. S.). The costs of publicationof this article were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

1 Departement de Biochimie, Faculte de medecine, Universite de Sherbrooke,Sherbrooke, Quebec J1H 5N4, Canada.

2 To whom correspondence should be addressed: Centre de recherche encancerologie, Hotel-Dieu de Quebec, 11 cote du Palais, Quebec G1R 2J6,Canada. Tel.: 418-525-4444 (ext. 15544); Fax: 418-691-5439; E-mail:[email protected].

3 The abbreviations used are: MT, metallothionein; ChIP, chromatin immuno-precipitation; EMSA, electrophoretic mobility shift assay; LUC, luciferase;MRE, metal regulatory elements; MTF-1, metal-responsive transcription

factor-1; tBHQ, tert-butylhydroquinone; C/EBP, CAAT/enhancer-bindingprotein; CMV, cytomegalovirus.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 13, pp. 8190 –8201, March 28, 2008© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

8190 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 13 • MARCH 28, 2008

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element protein-1, specifically binding with high affinity toMRE elements in a zinc-dependent manner, and subsequentlyshowed that this protein corresponds to MTF-1 thus support-ing the contention that MTF-1 is the only MRE-binding factor(23, 24). We also showed that purified MTF-1 binds to MREc,MREd, and MREe, as assayed by DNaseI footprinting analysis(23). More recently, in a model depicting the dynamic tran-scription factor complexes found at the proximal region of themouseMT-1promoter, it has been suggested thatMTF-1 occu-pies all MREs, including MREc, under metal-induced condi-tions (19). However, on the basis of other experiments carriedout in vitro and in vivo, it was suggested that NF1 interacts withthe MREc element, as well as with two other sites in theMREb region, and inhibits both constitutive and metal-in-duced MT gene transcription (25, 26). However, earlierreports from the same laboratory had concluded thatC/EBP� (or CP-1) (27) and CP-2 (28) bind to the MREcregion and activate MT transcription.In the course of our studies on the characterization of the

mouse MT-1 gene promoter, DNaseI footprinting analysesrevealed the presence of a nuclear protein, distinct fromMTF-1, binding to the MREc region. Given that the identity ofthe MREc-binding protein remains controversial, we furtheranalyzed this region. We show here that the MREc elementoverlaps with an atypical NF1 binding site and that a NF1-likeprotein binds in vitro to this region as well as to a second sitecontiguous with the Sp1a site. The NF1 protein contributespositively to the constitutive expression of the MT-1 gene andacts cooperatively with MTF-1 to activate MT gene transcrip-tion in response to metal ions and the phenolic antioxidanttert-butylhydroquinone (tBHQ). Amodel is presented inwhichzinc treatment inducesMTF-1-mediated alteration of chroma-tin structure, which allows the binding of positively acting fac-tors to theMT promoter.

EXPERIMENTAL PROCEDURES

Material—Restriction and DNA modifying enzymes wereobtained from New England Biolabs (Pickering, Ontario),[�-32P]dCTP was from (PerkinElmer Life Sciences), and syn-thetic oligonucleotides were from Invitrogen. The polyclonalanti-NF1 antibody used for the supershift was provided byReneSaint-Arnaud, Shriners Hospital, Montreal (29). All otherchemicals were purchased from Sigma.

Plasmid Constructs and Muta-genesis—Dr. Nicolas Mermodkindly provided the NF1 Rous sar-coma virus expression plasmidsp113-CTF-1 and p113-CTF-1�(30). Plasmid MT1-LUC (31) con-tains 1843 bp of the 5� flankingsequence and 68 bp of the 5�untranslated region from themouseMT-1 gene in the pGL2 basic vector(Promega, Madison, WI). Con-structs �-590-LUC, �-238-LUC,and �-150-LUC contain mouseMT-1 promoter sequence positions�590 (relative to the transcription

start point) to �68, �238 to �68, and �150 to �68, respec-tively, in pGL2 basic. To construct the reporter plasmids(NF1ab)MT1m-LUC and (MREdd)MT1m-LUC, the double-stranded oligonucleotides NF1ab and MREdd (Table 1),respectively, were inserted in front of a minimal mouse MT-1gene promoter (MT1m, �34 to �68) (31) in pGL2 basic. TheNF1ab oligonucleotides contain NF1a and NF1b sites orga-nized in tandem, whereas theMREdd oligonucleotide containstwo strong mouse MT-1 MREd elements in opposite orienta-tion. The reporter plasmid NF1ab(MREdd)MT1m-LUC wasgenerated by inserting the NF1ab oligonucleotide into plasmid(MREdd)MT1m-LUC upstream of the MREdd oligonucleo-tide. Plasmid (MREa)6MT1m-LUC contains six mouse MT-1MREa elements in front of aminimal mouseMT-1 promoter inpGL2 basic (32).Formutagenesis, a 1843-bp (-1843 to �68) MT1 promoter

fragment was subcloned into the plasmid pAlter-1 (Pro-mega), and mutations were introduced in different sitesusing specific oligonucleotides (Table 1) according to theinstructions of the manufacturer. The resulting fragmentswere subcloned into pGL2 Basic to generate a series of plas-mids with mutation(s) in the NF1a (NF1a�-LUC), NF1b(NF1b�-LUC), and NF1a and NF1b (NF1ab�-LUC) sites.For all the constructs and mutants, correct insertions andmutations were confirmed by sequencing.Cell Culture and Transfection—Metal-resistant mouse L50

fibroblast cells were obtained from Dean H. Hamer (NationalInstitutes of Health, Bethesda, MD). The human HepG2 cellsused for transient transfection were from A. Anderson (Centrede Recherche, l’Hotel-Dieu de Quebec), whereas those used togenerate stable transfectants were from Jacques Pouyssegur(Institute of Signaling, Developmental Biology and CancerResearch, Nice, France). For large scale nuclear extract prepa-rations, L50 cells were grown in suspension in Eagle’s minimalessential medium supplemented with 5% fetal calf serum and5%horse serum in the continuous presence of 50�MZnCl2. Forsmall scale analytic experiments, L50 and HepG2 cells werecultured inmonolayers in Dulbecco’s modified Eagle’s mediumsupplemented with 10% fetal bovine serum. Cells were trans-fected with the different plasmids by the calcium phosphatemethod (33), treated or not with CdCl2 (final concentration 2.5�M), ZnCl2 (100 �M), or tBHQ (100 �M) and harvested after6–8 h (32). Briefly, cells were seeded (�4 � 105/6-cm plate)

FIGURE 1. Map of the mouse MT-1 gene promoter. Schematic representation of the mouse MT-1 geneproximal promoter. Arrangement of the five MREs (arrows) (11, 12) is shown with the E-box 1 and the USF/AREelements (19), the binding sites for the transcription factor Sp1 and NF1, and the TATA box. Below the line, thecorresponding transcription factors interacting with the different elements are shown. The numbers at the toprefer to the positions relative to the transcription start point.

NF1 Activates the Mouse Metallothionein-1 Promoter

MARCH 28, 2008 • VOLUME 283 • NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 8191


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16 h prior to transfection. Cells were transfected with 1 �g ofthe reporter plasmidMT1-LUC, or the differentmutants, or 7.5�g of the plasmids (NF1ab)MT1m-LUC, (MREdd)MT1m-LUC, or NF1ab(MREdd)MT1m-LUC, shocked for 3 min at37 °C with 15% glycerol in HEPES-buffered saline, incubatedfor 12 h in growthmedium, and then treated or not withmetalsor tBHQ. In some experiments, cells were cotransfected with 1,5, 10, or 50 ng of the NF1 expression vectors p113-CTF-1 orp113-CTF-1�, as indicated in the figure legend. The plasmidpTK-rLUC (Promega) was used as internal standard tomonitortransfection efficiency. The total amount of DNA added to thecells was adjusted to 10 �g per dish with pGL2 basic DNA.Luciferase (LUC) activities were determined with a Dual-LUCassay kit (Promega) according to the recommendations of themanufacturer. The transcriptional activity of the reporter plas-mids was evaluated in duplicate in two or three different inde-pendent transfections.For stable transfections, HepG2 cells were cotransfected

with theMT1-LUC,NF1a�-LUC,NF1b�-LUC,NF1ab�-LUC,or pGL2-basic reporter plasmid, and the amiloride-resistantNa�-H� exchanger expression vector, pNHE1-R (kindly pro-vided by Dr. J. Pouyssegur) (34), with a 20:1 ratio. For the selec-tion of stably transfected cells, threeH�-suicide selectionswereapplied (34). Cell lysates were prepared from two differentpools of stable populations transfected with the different plas-mids. LUCactivitywasmeasured and expressed as relative LUCactivity permicrogramof total cellular protein. Protein concen-tration was determined by the Bradford method using a Bio-Rad assay kit (Mississauga, Ontario).Nuclear Extract Preparation, EMSAs, DNaseI Footprinting

Analysis, UV Cross-linking Assay, and Chromatography—Forthe NF1 EMSA, 5–6 �g of nuclear extract (24) was mixed inEMSA binding buffer (20 mM Tris, pH 7.6, 50 mM KCl, 2 mM

MgCl2, 3.3% Ficoll, 1 mM dithiothreitol, 0.1 mM phenylmethyl-sulfonyl fluoride) with 500 ng of poly[dI-dC]2 (GE Healthcare,Fairfield, CT) and 20 fmol of 32P-end-labeled probe, and themixture was incubated for 10 min at room temperature. Pro-tein-DNA complexes were subjected to polyacrylamide gelelectrophoresis in Tris/borate buffer (22 mM Tris base and 22mM boric acid). Methods for EMSA analysis using the C/EBPoligonucleotide as the probe have been described (24).DNaseI footprinting assays were performed (35) using a

mouse MT-1 restriction fragment 5�-end-labeled at �41 andextending to �200, or a mouseMT-1 PCR-amplified fragment,�348 to �72, as the probe (24). For the UV cross-linking assay(36), the probe was prepared by hybridizing the NF1b oligonu-cleotide (Table 1) to a 9-base complementary primer (5�-GCGTCCTT). This oligonucleotide was rendered completelydouble-stranded with he Klenow fragment of DNA polymeraseI in presence of [�32P]dATP. For competition experiments,specific double-stranded competitor oligonucleotides (Table1), as indicated in the figure legends, were added together withthe probe. In supershift experiments, 2 �l of an anti-NF1 or ananti-C/EBP-� (�198, Santa Cruz Biotechnologies, Santa Cruz,CA) polyclonal antibody was added to the binding reaction, themixture was incubated for 10min at 21 °C, followed by additionof the labeled oligonucleotide probe. Rabbit antiserum to

mouse MT-3 4 was used as a negative control. MRE-bindingproteins were purified from 60 ml of L50-cell crude nuclearextracts (10 �g protein/�l) by standard chromatography withNaCl gradient elution (24).Chromatin Immunoprecipitation Experiments—NF1 ChIP

assays were performed using the ChIP-it Express kit fromActive Motif (Carlsbad, CA) following the manufacturer’sinstruction. Cells were treated or not with 100 �M zinc for 3 hand then cross-linked with 1% formaldehyde. The chromatinwas immunoprecipitated with an anti-NF1 antibody (N-20X orH-300, Santa-Cruz Biotechnology, Inc.), or normal rabbitserumor IgG (Millipore, Billerica,MA). TheMTF-1ChIP assaywas performed as described (37) with somemodifications. Pro-tein A-Sepharose beads were first coupled to 6 �g of an anti-MTF-1 polyclonal antibody or preimmune serum in presenceof 20 �g of bovine serum albumin and 20 �g of herring spermDNA (Invitrogen). The Sepharose-conjugated anti-MTF-1antibody was then incubated overnight at 4 °C with an amountof chromatin corresponding to 1.6 � 106 liters of cells. Theresulting DNA was analyzed by PCR using a pair of primerscorresponding to the mouse MT-1 promoter region �230 to�80. As a negative control, each ChIP sample was also sub-jected to PCR using primers specific to a region located in thecoding region of the glucose-6-phosphate dehydrogenase gene(GenBankTM accession number X53617), positions �1841 to�1992. In some experiments, MTF-1-null mutant dko7 cells(38) (generously provided by Dr. W. Schaffner, Zurich) weretransfected with 500 ng of a CMV-MTF-1 expression vectors(24) 24 h before metal induction using the ExGen 500 transfec-tion reagent (Fermentas LifeSciences, Burlington, Ontario) fol-lowing the manufacturers’ instructions. PCR products wereseparated by agarose gel electrophoresis and visualized bySYBR Gold (Invitrogen) staining. Samples were subjected toPCR for different numbers of cycles to ensure that amplifica-tion was in the linear range. These ChIP experiments were per-formed three time using two different chromatin preparations.The anti-MTF-1 antibody was raised in rabbit by using puri-

fied, bacterially expressed protein representing the C-terminalregion of mouse MTF-1 (amino acids 577–675) fused to gluta-thione S-transferase. Anti-MTF-1 antibody was purified on aPROSEP-A column (Millipore, Etobicoke, ON). The anti-MTF-1 antibody specifically recognized a protein ofMr 100,000that is absent in dko7 cell extracts (data not shown).

RESULTS

Identification of a Protein Specifically Binding to the MREcRegion—To elucidate the nature of the MREc-binding protein,mouse nuclear extracts were fractionated on a heparin-Sepha-rose column and analyzed by DNaseI footprinting analysis. Inagreement with our previous observations (23, 24), footprintswere present over the Sp1a, MREd-Sp1b, MREc, and USF/AREsites (Fig. 2A, lane 1). MTF-1 binds to MREd and induces theformation of a DNaseI-hypersensitive site at �153 (Fig. 2A,lanes 4–6, stars). In heparin-Sepharose chromatography,MTF-1, defined as theMREd-binding protein, mainly eluted infractions 3–5, as indicated by the hypersensitive site at �153.

4 P. Moffatt and C. Seguin, unpublished results.




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However, the MREc-binding protein as well as the proteinbinding to the Sp1a region remained present in all the otherfractions, including fractions 11–13 depleted of MTF-1 (Fig.2A, compare lanes 4 and 14). This suggests the presence of twodistinct proteins. To assess the specificity of theMREc-bindingprotein, fractions 3–13 (Fig. 2A) were loaded onto an MREa-affinity column and eluted by three salt steps. Two MRE-bind-ing proteins species were eluted from theMRE affinity chroma-tography. First, an MREc-binding activity present in all thefractions from 250 mM to 650 mM salt and second, MTF-1found predominantly in the second 650 mM salt fraction (Fig.2B, lane 8, star). Like the MREc-binding protein, the Sp1a-binding protein was present in all the fractions (Fig. 2B),whereas USF/ARE activity was found in the 250 mM fraction(Fig. 2B, lane 3). Note that the 250 mM salt fraction containingtheMREc-binding protein is completely devoid ofMREd-bind-ing activity (Fig. 2B, lane 3), clearly indicating that the MREc-binding protein is distinct from the MREd-binding proteinMTF-1.To further characterize the MREc-binding protein, foot-

printing competition assays were performed using a crudenuclear extract (Fig. 3A) or the 250mM salt affinity fraction (Fig.3B) specifically containing the MREc-binding activity anddepleted of MTF-1 (see Fig. 2B, lane 3). The oligonucleotidesMREc and MREd (Table 1), as well as the negative controlMUTds (39), did not compete for the MREc-binding protein(Fig. 3, A and B). However, in MTF-1-containing extracts, theMREc andMREd oligonucleotides efficiently competedMTF-1on the MREd element (Fig. 3A, lanes 7–9, and data not shown)(36), thus further indicating that the MREc-binding protein isdistinct from MTF-1. Fine mapping of the footprint over theMREc region, performed by comparing with aMaxam and Gil-bert sequence ladder, revealed that the protected regionextends from �126 to �109 (Fig. 3C). Notably, this excludesthe first three nucleotides in the MRE consensus sequence ofthe MREc element, namely the highly conserved TGC nucleo-tides. Because each of these three nucleotides is critical formetal induction (40) and MTF-1 DNA binding (41), this virtu-ally excludes the possibility that theMREc-binding protein cor-responds to MTF-1. This also suggests that the MREc elementis not a bona fideMRE.Close examination of the nucleotide sequence of the pro-

tected region using DNA transcription factor binding site pre-diction programs identified a perfect NF1 half site (GCCAA,NF1b) in the protected region. In addition, a second NF1 halfsite (NF1a) is present on the mouseMT-1 promoter upstreamof the Sp1a site (Figs. 3C and 4A). To further assess the identityof theMREc-binding protein, DNaseI footprinting competitionexperiments were performed using as competitor DNA an oli-gonucleotide, MREcl, corresponding to the DNaseI protectedregion in the MREc region, namely the entire MREc consensussequence and the putative NF1 half site (Fig. 3C). We also usedthe oligonucleotides NF1b, a shorter version ofMREcl in whichthe first nucleotide of theMREc core sequence is excluded (Fig.3C), MT3-SN (31), an oligonucleotide corresponding to theoverlapping Sp1/NF1 site of the mouse MT-3 promoter, Sp1a,corresponding to the Sp1a site, and NF1, an oligonucleotidecontaining a generic NF1 site (Santa Cruz Biotechnologies). As

FIGURE 2. Identification of two distinct MRE-binding proteins. A, DNa-seI footprinting analysis performed with chromatographic fractions fromthe heparin-Sepharose column eluted with a NaCl gradient, as indicatedschematically over the lanes. Note that the MREc-binding activity is pres-ent in all the fractions, whereas the MREd-binding activity is mainly pres-ent in fractions 3–5, as evidenced by the DNaseI hypersensitive site (stars).This hypersensitive site is generated by the binding of MTF-1 on MREd.B, DNaseI footprinting analysis performed with fractions from the MREaffinity column eluted with a NaCl step gradient, as indicated. Fraction 4(lane 3) from the 250 mM salt fraction contains a specific MREc-bindingactivity, whereas MTF-1 mainly eluted in the second 650 mM salt fraction(lane 8). The probe was a mouse MT-1 gene promoter DNA fragmentextending from �200 to �41. The positions of the different cis-actingelements are indicated on the left, as determined by Maxam-Gilbertsequencing. Numbers above the lanes indicate the fraction numbers,whereas those below the gel correspond to the lanes. Lanes 2 and 3 on eachpanel are non-adjacent lanes from the same gel. Lanes: L50, L50-cellnuclear extract; FT, flow-through; No, no extract.




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shown in Fig. 3B,MREcl efficiently competed the footprint overthe MREc region, thus indicating that the nucleotides requiredfor binding of theMREc-DNAbinding protein are distinct fromthose required for MTF-1 and are located downstream of theMRE consensus sequence in the NF1 half site. Most interest-ingly, MREcl also competed for a protein binding the NF1 por-tion of the footprint in the NF1a-Sp1a region (Fig. 3B). In fact,in vivo footprinting assays suggested that an unknown factor(s)interacts with this region (17). This protein is distinct fromSp1,because the Sp1a oligonucleotide did not compete theNF1 por-tion of the Sp1a-NF1a footprint but could efficiently competeits downstream Sp1 portion (Fig. 3,A andB). Similarly, theNF1andNF1b oligonucleotides competed for the protein(s) bindingto the MREc region and the NF1 portion of the NF1a-Sp1afootprint (Fig. 4), whereas the oligonucleotide MT3-SN or amixture of Sp1a and NF1 oligonucleotides competed for thefootprint over the MREc region as well as the entire footprintover the NF1a/Sp1a region (Fig. 4). These results strongly sug-gest that the MREc-binding protein corresponds to NF1, or aclosely related family member, and that this protein interactswith a second site on themouseMT-1 promoter, namely NF1a,adjacent to the Sp1a site.NF1 Binds to the Mouse MT-1 Promoter—To obtain an indi-

cation of the molecular weight of the MREc-binding protein,UV cross-linking experiments were performedwith crude L50-cell nuclear extracts and different chromatographic fractions.

FIGURE 3. Competition experiments in DNaseI footprinting assays.A, footprinting reaction was carried out with L50-cell crude nuclear extracts,and competition was performed with double-stranded unlabeled oligonu-cleotides (Table 1) corresponding to the mouse MT-1 promoter Sp1a site(Sp1a), the mouse MT-1 MREd (MREd), and the nonspecific oligonucleotideMUTds (39). The probe was the same as in Fig. 2. Lanes 6 and 7 are non-adjacent lanes from the same gel. B, footprinting reaction was carried outwith aliquots of fraction 4 (Fig. 2B, lane 3) of the heparin-Sepharose columncontaining the specific MREc-binding activity. Competition was performedwith double-stranded unlabeled oligonucleotides corresponding to an

extended region of the mouse MT-1 promoter around MREc (MREcl), themouse MT-1 MREc (MREc), and the oligonucleotides MUTds and Sp1a. Theprobe was a mouse MT-1 gene promoter DNA fragment extending from�348 to �72. 20 –100 ng of competitors, as indicated above the lanes, wereadded together with the probe, and binding was allowed to proceed for10 –15 min at 24 °C, before adding the DNaseI. Lanes: 0, no competitor; L50,L50-cell nuclear extract; No, no extract. The positions of the different cis-act-ing elements are indicated on the left as determined by Maxam-Gilbertsequencing. Numbers below the gel correspond to the lanes. C, sequence ofthe mouse MT-1 promoter, nucleotides �212 to �99, encompassing the twoNF1 sites. MREs and Sp1 sites are underlined, and the NF1 sites are boxed. Thefootprints present in this region are indicated over the sequences, and theoligonucleotides used as competitors are listed under the sequence.

TABLE 1Sequences of the synthetic oligonucleotides used in this studyNucleotides in bold designate the coreMRE sequence, those in bold and underlinedare theNF1 core conserved sequences, and those in italic and underlined are the Sp1site. Nucleotides in lowercase letters at the extremities of the oligonucleotides cor-respond to restriction sites added to the oligonucleotides, whereas those in themiddle of the oligonucleotides correspond to mutations introduced in a given site.In NF1ab, the NF1a (bold) andNF1b (underlined) sites are shown.MREdd containstwo strong mouseMT-1MREd in opposite orientation (bold and underlined). TheNF1 oligonucleotide is from Santa Cruz Biotechnologies.

Name SequenceNF1 5�-TTTTGGATTGAAGCCAATATGATAANF1a 5�-CCGAGCCAGTCGTGCCAAAGGNF1b 5�-GCGCTCGGCTCTGCCAAGGACGCNF1ab 5�-tcgagATCCGAGCCAGTCGTGCCAAAGGGCGCTC

GGCTCTGCCAAGGACGCMREc 5�-gatccAAAGTGCGCTCGGCTCaMREcl 5�-gatcccAGTGCGCTCGGCTCTGCCAAGGACGCaMREd 5�-cgatCTCTGCACTCCGCCCGAMREdd 5�-tcgAAGATCTCGGGCGGAGTGCAGAGCTAGCTC

TGCACTCCGCCCGAcSp1a 5�-gatccAAGGGGCGGTCCCGCaC/EBP 5�-TGCAGATTGCGCAATCTGCANF�B 5�-CAACGGCAGGGGAATCTCCCTCTCCTTNF1a� 5�-CGAGCCAGTCGTtaaAAAGGGGCGGTCNF1b� 5�-GCGCTCGGCTCTtaaAAGGACGCGGGG




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The major protein species complexed with the NF1b oligonu-cleotide migrated on a denaturating gel with an apparentMr of�60,000 (Fig. 5, lane 2), and this complex was enriched in theheparin-Sepharose fraction 9 and the affinity chromatographyfraction 3, which contains high levels of MREc-binding protein(Fig. 5, lanes 4 and 6). No significant labeled species was gener-ated in chromatographic fractions devoid of MREc-bindingactivity, namely the heparin-Sepharose and the affinity chro-matography column flow-through (Fig. 5, lanes 3 and 5). Inclu-sion of a 250-fold excess of cold MREcl oligonucleotide com-pletely abolished complex formation (Fig. 5, lane 7). Becausethe covalent attachment of short oligonucleotides to proteinshas only a minor effect on the mobility of these proteins inSDS-PAGE, these experiments indicate that a protein of �60kDa binds to the MREc region. The molecular mass of thisprotein is consistent with that of NF1 (50–70 kDa).The evidence discussed above strongly suggests that the

MREc-binding protein is NF1 or a closely related factor. Tofurther ascertain the identity of the MREc-binding protein,EMSA analyses were performed using 32P-labeled NF1b oligo-nucleotide as the probe. Because a C/EBP-related protein hasbeen reported to bind to the mouse MT-1 promoter regions

around the NF1a and NF1b sites (27), EMSA analyses wereperformed using HepG2 cell nuclear extracts that contain bothNF1 andC/EBPprotein species (42, 43). Incubation of theNF1boligonucleotide with HepG2 cell nuclear extracts led to the for-mation of two major complexes (Fig. 6A, lane 1). Competitionexperiments were performed to address the specificity of thebinding. Consistent with the DNaseI footprinting data, EMSAanalysis showed that the complexes formed with the NF1b oli-gonucleotide and HepG2 cell nuclear proteins were efficientlycompeted by the NF1, NF1a, NF1b, and MREcl oligonucleo-tides, whereas the MUTds, C/EBP, MREc, MREd oligonucleo-tides (Fig. 6A), as well as oligonucleotides corresponding toSTAT and NF-�B binding sites (data not shown), did not com-pete. These competition data further confirm that the MREc-binding protein corresponds to NF1 and not to C/EBP. Toobtain more conclusive evidence, we analyzed the MREc pro-tein by supershift assays using a polyclonal anti-NF1 antibody.HepG2 cell nuclear extracts were incubated with NF1 anti-serum or with an anti-C/EBP or an anti-MT3 polyclonal anti-body prior to incubation with the NF1b oligonucleotide in theEMSA. Incubation with the NF1 antiserum but not with theMT3 or the C/EBP antiserum resulted in the complete elimina-tion of the two major complexes and the formation of a super-shifted complex (Fig. 6A, lane 10, and data not shown). Thespecificity of the supershifted complex was demonstrated bythe lack of a similar effect of the same NF1 antibody on com-plexes formed between oligonucleotide C/EBP and HepG2 cellnuclear extracts (Fig. 6B, lane 5). The presence of C/EBP in the

FIGURE 4. Identification of two NF1 half sites in the mouse MT-1 pro-moter. A, alignment of the two MT-1 NF1 sites with NF1 (45) and C/EBP (61)consensus sequences. Underlined nucleotides correspond to nucleotidesconserved in the mouse MT-1 NF1 sites. B, competition experiments in DNaseIfootprinting assays. The footprinting reaction was carried out as described inFig. 3B, using as competitors double-stranded unlabeled oligonucleotidescorresponding to the mouse MT-1 NF1b site (NF1b), a generic NF1 site (NF1),the mouse MT-3 promoter NF1/Sp1 site (SN), and the oligonucleotide Sp1aalone (Sp1) or in combination with the oligonucleotide NF1b (Sp1�NF1).

FIGURE 5. Identification of the NF1b DNA-binding activity as a 60-kDapolypeptide by UV cross-linking assay. DNA affinity labeling of NF1b-bind-ing factors in a L50-cell crude nuclear extract (L50), the flow-through of theheparin-Sepharose column (FT1), fraction 9 (9) of the heparin-Sepharose col-umn enriched with the MREc-binding protein, the flow-trough (FT2) of theMRE affinity column (MRE affinity), and fraction 3 of the affinity column spe-cifically containing the MREc-binding activity are shown. The probe was theNF1b oligonucleotide (Table 1). Addition of cold MREcl oligonucleotide (cl) tothe reaction completely inhibited formation of the �60-kDa DNA-proteincomplex (arrow). Numbers below the gel indicate the lanes. The asterisk indi-cates nonspecific binding. Lanes: M, molecular weight markers; 0, no compet-itor. F, free probe.




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HepG2 extract was ascertained by performing an EMSA usingtheC/EBP oligonucleotide as the probe. As shown in Fig. 6B thecomplexes formed with the C/EBP oligonucleotide are effi-ciently competed with cold C/EBP DNA but not with theMUTds oligonucleotide.Moreover, treatmentwith anti-C/EBPantiserum generated a supershift (Fig. 6B, lane 4), whereastreatment with anti-NF1 antibody did not. This clearly showsthat theHepG2 cell extracts containC/EBPprotein and that thebinding of NF1 to the NF1b oligonucleotide in Fig. 6A was notartificially favored because of the absence of C/EBP protein inthe extracts. Taken together, these results strongly support theconclusion that NF1 is the MREc-binding protein.NF1 Activates theMouseMT-1 Promoter—To determine the

functional effect of NF1 on constitutive and metal-inducedactivity of the mouseMT-1 promoter, HepG2 cells were trans-fected with the reporter plasmid MT1-LUC or various NF1mutants. We confirmed that mutation of the NF1 sites abro-gated binding of NF1 proteins by DNaseI footprinting analysesusing the corresponding mutant promoter fragments as theprobe (data not shown). Cells transfected with the wild-typecontrol MT1-LUC reporter plasmid displayed a basal LUCactivity that was induced 20- and 80-fold by the addition ofZnCl2 and CdCl2, respectively (Fig. 7A). Mutation of the NF1asite in the context of the intactMT promoter fragment dimin-ished basalMT promoter activity by 60% and zinc- and cadmi-um-induced levels by �40 and 55%, respectively. However, themutant promoter remained strongly inducible andwas induced30- and 100-fold in response to zinc and cadmium, respectively.Similarly, inactivation of the NF1b site led to a 50% decrease ofbasal LUC activity and to a 35 and 40% reduction of zinc- andcadmium-induced promoter activity. The NF1b mutant wasstill induced 30- and 100-fold in response to zinc and cadmium,

respectively. For the double mutantNF1ab, basal levels were inhibitedby 55%, whereas zinc and cadmiuminduction was reduced by 55 and70%, respectively. Promoter activityof the double mutant was induced20- and 60-fold by zinc and cad-mium, respectively.To further confirm the function

of the NF1 sites in stimulating pro-moter activity, we used deletionmutants in transfection experi-ments. As reported earlier (11, 12),deletion of the mouse MT-1 pro-moter sequences between �1843and �590, or �238, did not sub-stantially modify either basal ormetal-induced expression (Fig. 7B).However, further deletion to �150produced a 40% decrease in basallevel but had only a marginal effecton the capacity of the promoter tobe induced by metal ions. Com-pared with 20-fold for the �1843control reporter plasmid, the �150deletion mutant was induced �15-

fold.However, similar to theNF1a orNF1bmutants, the overallactivity of the promoter was reduced by �50%. This showedthat the region between �238 and �150, which includes theNF1a, E-box1, and Sp1a sites, contains elements required forthe basal level of expression of the gene and maximal metalinduction. These results are in congruence with those obtainedwith the NF1 mutants. Overall, these results indicate that,although the NF1 sites are not essential, as expected, to confermetal induction to themouseMT-1 promoter, their presence isrequired formaximal constitutive andmetal-induced promoteractivity.To substantiate the stimulating effect of NF1 on MT gene

transcription, the MT1-LUC or the double NF1 mutantMT1(NF1ab)�-LUC reporter plasmids was cotransfected intoHepG2 cells along with the NF1 expression plasmid, p113-CTF-1 or the transcriptionally inactive mutant p113-CTF-1�,in presence or in absence of inducers. Cotransfection of thewild-type NF1 expression vector had no effect on either consti-tutive or metal-induced expression levels of both wild type andmutant MT promoters (Fig. 7C). However, cotransfection ofthe dominant negative mutant led to a dose-dependent inhibi-tion of metal-induced MT promoter expression (Fig. 7D) buthad no effect on basal levels. At the highest doses (10 and 50 ng),transfection of p113-CTF� reduced zinc-stimulated LUC activ-ity by 50%. These results further support a positive regulatoryrole of NF1 onMT gene transcription.To determine whether NF1 acts synergistically with MTF-1

in the activation of the MT-1 promoter in response to metalions, we constructed synthetic promoters containing NF1 andMRE sites isolated from other cis-acting elements. These plas-mids contain a minimal mouse MT-1 promoter joined to twoMREds (MREdd), the NF1a and NF1b sites (NF1ab), or a com-

FIGURE 6. The NF1 transcription factor present in nuclear extracts specifically binds to the NF1b oligo-nucleotide. A, EMSA reactions were performed by incubating 32P-labeled NF1b oligonucleotide (20 fmol, �0.5ng), with HepG2-cell nuclear extracts. For competition reactions, 100 ng of double-stranded unlabeled oligo-nucleotides (Table 1) were used as indicated above the lanes. B, reactions were performed using the 32P-labeled C/EBP oligonucleotide as the probe and a HepG2-cell nuclear extract. Competition was performed withdouble-stranded unlabeled C/EBP and MUTds oligonucleotides. Anti-NF1 (Ab-NF1), anti-C/EBP (Ab-C/EBP) anti-bodies or nonspecific anti-MT-3 antibody (Ab-MT-3) were added to the EMSA reactions, and the mixtures werepreincubated for 5 min at 21 °C before the addition of 32P-labeled oligonucleotide probes. Arrows indicate theDNA-protein complexes. SS refers to supershifted complexes. F, free probe; B, bound DNA.




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bination of both theMREd elements and the NF1 sites (NF1ab/MREdd). HepG2 cells transfected with the NF1ab plasmidshowed low constitutive activity that did not significantlyincrease in presence of metals (Fig. 7E). In contrast, cells trans-fected with MREdd showed a basal transcription level twice ashigh as that of NF1ab that was induced 2.7- and 3.6-fold by zinc

and cadmium, respectively. Inter-estingly, the fusion promoterNF1ab/MREdd was slightly moreactive in the non-induced state andwas induced 4- and 5-fold by zincand cadmium, respectively. Theseresults showed that NF1 acts syner-gistically withMTF-1 formetal acti-vation of MT gene transcription.This synergy between these twotranscription factors is even moreclearly observed in stable transfec-tants (see below).Because correct chromatin struc-

ture, which cannot be achieved insmall transiently expressed plas-mids, may significantly alter geneexpression by affecting interactionsof transcription factors with thepromoter, we confirmed the posi-tive role of NF1 onMT gene expres-sion by generating stable transfec-tants in HepG2 cells with the sameplasmids used in transient transfec-tion experiments. Two pools ofclones generated from two distincttransfections for each constructwere analyzed. The results obtainedwith the stable transfectants are ingood agreement with those ob-tained in transient transfections.Indeed, mutation of the NF1 sitesreduced basal and metal-inducedtranscriptional activity of the MTpromoter by 40–90% (Fig. 8A). Asobserved in transiently transfectedcells, the NF1 mutants were stillstrongly inducible in response tometals, between 5- and 10-fold. TheNF1 mutations also diminishedinduced transcription levels inresponse to the phenolic antioxi-dant tBHQ, a known MT geneinducer (9, 44) (Fig. 8A). The induc-tion of MT by tBHQ requiresMTF-1 (44). However, the moststriking result was obtainedwith thesynthetic promoters. The NF1abreporter plasmid displayed lowbasal transcription levels in stabletransfected cells and was not metal-nor tBHQ-inducible. The MREdd

and (MREa)6 reporters were 3- to 6-fold induced by metals andweakly inducible by tBHQ. Notably, the presence of both MREand NF1 sites strongly increased basal and metal- and tBHQ-inducible transcriptional activity (Fig. 8B). These results clearlyshow the importance of NF1 for the optimal activation of MTgene transcription by metal ions and tBHQ.

FIGURE 7. Transient transfection studies in HepG2 cells. A, cells transfected with a plasmid mixture, includ-ing the reporter wild-type MT1-LUC (WT, 1), containing 1843 bp of mouse MT-1 gene 5� flanking sequence, orthe NF1 mutant reporter plasmids NF1a�-LUC (NF1a�, 3), NF1b�-LUC (NF1b�, 2), or NF1ab�-LUC (NF1ab�, 4)and pTK-rLUC, as internal standard, were treated or not with 100 �M ZnCl2 or 2.5 �M CdCl2 for 6 – 8 h. Cellextracts were prepared and LUC activity was measured with a dual LUC kit. Results are expressed as percentageof firefly LUC (fLUC) activity relative to the level directed by the renilla LUC (rLUC) construct, and as a percentagerelative to that of the WT DNA induced by zinc, which is taken as 100. Inset: basal levels plotted on a differentscale. Data represent the average � S.D. of three independent experiments performed in duplicate or intriplicate. B, cells were transfected as described in panel A with a plasmid mixture containing MT1-LUC (WT) or5� deletion mutant reporter plasmids, as indicated, and pTK-rLUC. C, cells were transfected as described inpanel A with a plasmid mixture of MT1-LUC (WT) or the NF1 double mutant NF1ab�-LUC (NF1ab�), the internalstandard pTK-rLUC, and increasing amount of the NF1 expression vector p113-CTF-1 (CTF1), as indicated.D, cells were transfected as described in panel A with a plasmid mixture containing MT1-LUC, the internalstandard pTK-rLUC, and increasing amount of the NF1 mutant expression vector p113-CTF-1�, as indicated.E, cells were transfected as described in panel A with LUC reporter plasmids containing the mouse MT-1minimal promoter (�35 to �68) fused to two MREd elements (MREdd), the NF1a and NF1b sites (NF1ab), or acombination of both the NF1 sites and the double MREd element (NF1ab/MREdd). Results are expressed aspercentage of firefly LUC (fLUC) activity relative to the level directed by the Renilla LUC (rLUC) construct, and asa percentage relative to that of MREdd plasmid induced by zinc, which is taken as 100. pGL2 (5 in inset),pGL2-Basic plasmid.




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An important question concerns the mechanism by whichNF1 potentiates the activation of MT gene transcription inresponse to metals and tBHQ. In fact, these results do notaddress whether NF1 andMTF-1 co-occupy theMT promotersimultaneously in response to metal induction or whether thebinding of NF1 requires first the binding of MTF-1. Thus, tobetter understand the mechanisms by which NF1 affects MTgene transcription, we address directly whether binding of NF1to the mouse MT-1 promoter is modulated by metal andwhether the presence ofMTF-1 is required for NF1DNA bind-ing by using the ChIP assay, a method that allows to study thedynamic in which transcription factors interact with DNA invivo in the context of the intact chromatin. First, to validate ourChIP assay, we examined the interaction of MTF-1 with theMT-1 promoter in L cells. As previously shown (19), MTF-1bound to theMT promoter in uninduced cells and this bindingis strongly enhanced in presence of zinc (Fig. 9, lower panel,lanes 1 and 2). ChIP assays of chromatin fromMTF-1-null dko7cells confirmed the specificity of the immunoprecipitation (Fig.9, lower panel, lanes 3 and 4). Second, we studied the binding ofNF1 to theMT promoter. In agreement with the in vitro stud-ies, the ChIP assay showed that NF1 binds to theMT promoterin vivo in L cells but not to the glucose-6-phosphate dehydro-genase gene coding region used as negative control (Fig. 9,upper panel, lane 1). Notably, zinc treatment strongly stimu-

lated NF1 DNA binding to the MT promoter (Fig. 9, upperpanel, lane 2). In dko7 cells, constitutive NF1 DNA-bindingactivity was lower than in L cells and was not metal-inducible(Fig. 9, upper panel, lanes 3 and 4). Most interestingly, metal-induction of NF1 DNA binding activity was completelyrestored in dko7 cells by expressingMTF-1 (Fig. 9, upper panel,lane 5). These results indicate that MTF-1 enhances NF1 DNAbinding to the MT promoter in the uninduced state and showthat MTF-1 is essential for the induction of NF1 DNA bindingin response to metals.

DISCUSSION

NF1 Binds to and Activates the Mouse Metallothionein-1Promoter—We have performed in vitro footprinting analysesand showed that an NF1 family member, or a closely relatedfactor, binds to theMREc region nucleotides�126 to�105 andto a second site, �205 to �187, upstream of the Sp1a site. Fivekinds of evidence support this conclusion. First, they contain aperfect NF1 half site (GCCAA). NFI protein binds as a dimer tothe dyad symmetric consensus sequence TTGGC(N5)GCCAAon duplex DNA (45). However, NF1 could also bind specificallyto individual half sites (TTGGC or GCCAA). In fact, both pro-tected regions in the footprint experiments include a NF1 halfsite (Fig. 3C). Moreover, the sequence TCG(N5)GCCAA in thepromoter of the �2 (I) collagen promoter is identical to theNF1a site of the mouse MT-1 promoter and binds NF1 (46).Second, that this factor is NF1 is further demonstrated by com-petition experiments using a fragment of themouseMT-3 pro-

FIGURE 8. Stable transfection studies in HepG2 cells. A, cells stably trans-fected with the MT1-LUC (WT), NF1a�-LUC (NF1a�), NF1b�-LUC (NF1b�),NF1ab�-LUC (NF1ab�), or pGL2-basic (not shown) reporter plasmids, weretreated or not with 100 �M ZnCl2, 2.5 �M CdCl2, or 100 �M tBHQ for 6 – 8 h. Cellslysates were prepared from two different pools of stable transfectants, andLUC activity was measured and expressed as relative fLUC activity per micro-gram of total protein. The results of one pool are shown. B, cells were stablytransfected with synthetic promoters containing the mouse MT-1 minimalpromoter fused to two MREd elements (MREdd), six MREa elements ((MREa)6),the NF1a and NF1b sites (NF1ab), or a combination of both the NF1 sites andthe double MREd element (NF1ab/MREdd). Cell lysates were prepared, andLUC activity was measured as described in panel A.

FIGURE 9. NF1 binds the mouse MT-1 promoter in vivo in a zinc-inducibleand MTF-1-dependent manner. ChIP assays were performed using chroma-tin isolated from L cells or dko7 mouse embryonic fibroblasts (MTF-1 null)treated or not with 100 �M ZnCl2 for 3 h prior to formaldehyde cross-linking.Immunoprecipitation of cross-linked chromatin was done with NF1 (upperpanel), MTF-1 (lower panel) antibodies or a pre-immune normal rabbit serum(PI), as indicated. DNA from both the immunoprecipitation input and theimmunoprecipitation-bound fractions was amplified by PCR with primerpairs for the mouse MT-1 promoter (MT-1) and the coding region of the glu-cose-6-phosphate dehydrogenase gene (G6PD). Some dko cells were trans-fected with a MTF-1 expression vector (CMV-MTF-1), grown for 24 h, and thentreated with zinc. Input: amplification of DNA prior to immunoprecipitation.The input sample contained 0.4% of the supernatant used for NF1 immuno-precipitation and 0.8% for MTF-1. The PCR products were analyzed by agar-ose gel electrophoresis. These ChIP assays were performed three times withsimilar results using two different chromatin preparations.




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moter that contains a NF1 binding site as well as a commercialoligonucleotide containing a consensus sequence forNF1 bind-ing sites. Third, UV cross-linking experiments indicate that aprotein of �60 kDa, consistent with that of NF1 (50–70 kDa)(45), binds to the MREc region. Fourth, an anti-NF1 antiserumrecognized the complexes formed in vitro between HepG2nuclear proteins and the NF1b oligonucleotide in supershiftexperiments (Fig. 6A, and data not shown), and fifth, ChIPassays showed that NF1 binds in vivo to the MT-1 proximalpromoter in a metal- and MTF-1-dependent manner.Our results do not support the contention that C/EBP�

(C�BP-1) (27) or C�BP-2/CP2 (28) binds to the NF1b site. First,EMSA competition experiments using an oligonucleotide con-taining a consensus C/EBP site did not prevent the binding ofnuclear proteins to the NF1 probe. Second, anti-C/EBP anti-body failed to disrupt the formation ofDNA-protein complexes(data not shown). The potential role of C/EBP on MT geneexpression, if any, appears to be inhibitory rather than stimula-tory, because stable expression of C/EBP� in prostate cancercells down-regulates MT gene expression (47). The molecularmass of C�BP-2/CP2 is 28 kDa, whereas that of the proteininteracting with the NF1 oligonucleotide is �60 kDa. Hence itis most unlikely that the protein detected in this study corre-sponds to C�BP-2/CP2. The reasons of these apparent discrep-ancies are not clear but could be related to cell-type differences.The NF1 family of site-specific DNA-binding proteins (also

known as CTF or CAAT box transcription factor) is composedof four members in vertebrates (NFI-A, NFI-B, NFI-C, andNFI-X).NF1 genes are differentially spliced, yielding asmany asnine distinct proteins from a single gene. The products of thefour NF1 genes differ in their abilities to either activate orrepress transcription (45). To determine the role of the NF1sites in MT gene transcription, transfection experiments wereperformed in HepG2 cells with a MT1-LUC reporter plasmidand specific mutants in which the NF1 sites were inactivatedindividually or together. Inactivation of either of the NF1 twosites diminished basal and metal-induced transcription rate ofthe MT-1 promoter by 35–60% in transient transfections,whereas inactivation of both sites led to a 55–70% inhibition.This inhibition was even more pronounced in stable transfec-tants in which transcriptional activity in NF1 mutants wasreduced by up to 90%. However, both in transient and stabletransfectants, metal induction, i.e. the ratio of basal overinduced transcription levels, was largely unaffected. Deletionmutant studies support the idea that the NF1 sites are positive-regulatory cis-acting elements. Indeed, in agreement with ear-lier studies (11, 12), deletions of the promoter region encom-passing the NF1a sequences significantly reduced both basaland metal-induced transcriptional activity of the MT-1 pro-moter. Moreover, experiments with synthetic promoterreporter plasmids containing NF1 orMRE sites or both regula-tory sequences fused to a minimal mouse MT-1 promotershowed that a much higher constitutive and metal-inducedactivity was observed with the reporter plasmids containingboth theMRE and theNF1 sites (Figs. 7E and 8B). Overall, theseresults clearly show thatNF1 is a positive regulator of both basalandmetal- and tBHQ-inducedMT transcription, acts synergis-tically with MTF-1 to activate the mouse MT-1 promoter in

response to metal ions and tBHQ, and contributes to maximalactivation of the gene.However, other studies have indicated a negative effect of

NF1 on MTF-1-mediated transactivation (25, 48). In thosestudies, transfection of vectors expressing NF1 proteins inHepG2 cells inhibited transcription from the MT or MTF-1reporter plasmids. Under our experimental conditions, expres-sion of wild-type NF1 did not affect MT1-LUC transcriptionmost likely because NF1 proteins are abundant and presentinside the cell in saturating concentration. However, expres-sion of the transcriptionally inactive mutant p113-CTF-1�reduced zinc-inducedMT1 promoter activity by up to 50% in adose-dependent manner (Fig. 7D), thus supporting the ideathat NF1 is a positive regulator of MT gene transcription.Indeed, if NF1 played a negative regulatory function on MTgene expression, the inactivemutant would have been expectedto lead to an increase in the transcriptional activity of thereporter plasmid. The reason for this apparent discrepancy isnot clear, but it may reflect the fact that NF1-mediated inhibi-tion ofMTpromoter transcriptionwas observed in transfectionexperiments using a strong CMV expression vector and 30- to1000-fold more vector than in this study, that is 1500 (25) and5000 (48) ng, compared with 5–50 ng. NF1 is an abundant,constitutively expressed, and ubiquitous transcription factor.Increasing too much its concentration could interfere withsome cellular components of the transcription machinery andindirectly compromise or quench MTF-1 activity. The strongCMV promoter can drive high levels of transcription leading topotentially non-physiological concentrations of the corre-sponding protein. Consequently, by using a weaker Rous sar-coma virus expression vector and by keeping the amount oftransfected plasmidDNA in the lower nanogram range, wemayhave avoided possible non-physiological effects.Proposed Mechanism—Using the CASTing method to iden-

tify MTF-1 binding motifs, several strong consensus sequencesfor NF1 were determined (48). These NF1 sequences did notbind MTF-1, thus suggesting that MTF-1 and NF1 may physi-cally interact. However, co-immunoprecipitation analysesusing NF1 andMTF-1 antibodies failed to demonstrate a directinteraction between these two proteins.5

The molecular mechanisms by which metals exert theiraction onMTF-1 are only partially understood. It has been pro-posed that MTF-1 act as positive regulator in the presence ofzinc ions by undergoing conformational changes that promoteDNA binding and transcription, thus facilitating the recruit-ment of other components of the transcription machineryincluding other transcription factors or cofactors (6, 7). In fact,TFIID and the Mediator complex interact functionally in aMTF-1-dependent manner to modulate transcriptionalresponse to metal ions (49). Phosphorylation is also involved inthe activation of MTF-1 (32). Several other proteins interactwith the mouseMT-1 promoter, including USF-1, USF-2 (15–19), Sp1 (9, 19–21), c-Fos (19), and c-Jun (9). These transcrip-tion factors may be essential for maintenance of adequate basalpromoter activities and for maximum induction in response to

5 G. St-Gelais and C. Seguin, unpublished results.




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inducers (8). For example, MTF-1 and USF1 cooperate to reg-ulate mouse MT-1 expression in response to zinc, and loss ofUSF-1 attenuatesMT gene expression (18). An important ques-tion then concerns the mechanism by which basal elementsamplifyMTF-1-mediatedmetal treatment.One possiblemech-anism is that MTF-1 stabilizes or allows the binding of one ormore factors to an adjacent enhancer element.We propose a model based on the induction of the mouse

mammary tumor virus promoter by glucocorticoids and ofcytochrome P450 1A1 by polycyclic aromatic hydrocarbons(50, 51). In both cases, activation of the respective genes by theligand results in binding of NF1 to its site, presumably becauseof changes in chromatin structure. Notably, the affinity of NF1for its DNA site is greatly affected by specific chromatin orga-nization (52). We speculate that, in addition to recruit TFIID,MTF-1 controls metal-mediated MT gene induction in partthrough the modification of chromatin structure and the sub-sequent recruitment of NF1 to theMT-1 promoter. Consistentwith this model, we showed here using ChIP assays that NF1binds in vivo to theMT-1 promoter in a metal- andMTF-1-de-pendent manner. Thus, NF1 binding to DNA would largely beinhibited due to a “closed” chromatin structure. Upon metalinduction, binding ofMTF-1 toMREd andMREawould inducea more “open,” accessible, chromatin structure, thus allowingthe binding of NF1 to the MT promoter. Consistent with thishypothesis, theMREc region encompassing theNF1b site of themouse MT-1 promoter shows metal-induced protection in invivo footprinting assays (9, 21). Although the interaction ofc-Jun,USF-1,USF-2, and Sp1with theMT-1promoter ismetal-and MTF1-independent, the recruitment of c-Fos requiresMTF-1 (19) and could also be recruited to the promoter withNF1. The formation of a DNaseI-hypersensitive site at the levelofMREd in presence of nuclear extract (Fig. 2,A andB, and datanot shown) provides some evidence of a conformational changein the DNA. In fact, it has been shown that the binding ofMTF-1 induces conformational changes in the MREd (53).Changes in chromatin structure of the MT-1 gene after metalinduction was also detected by general DNaseI I sensitivity (54,55). This model is also consistent withMTF-1 acting as a chro-matin insulator shielding specific transcriptionally activeregions from the repressive effects of flanking chromatin (56).Mutation of the NF1 sites did not only impair metal induc-

tion but also reduced constitutive expression thus suggestingthat NF1 binds to DNA in the absence of inducers. If, as sug-gested here, NF1 DNA binding is MTF-1-dependent, it followsthat MTF-1 also binds to DNA in basal conditions. In fact,MTF-1 is absolutely required for basal level transcription ofMTgenes (22). Serum-supplemented medium contains 3–4 �Mzinc, and this concentration is sufficient to drive MTF-1-de-pendent expression of the MT-1 gene (19). A significantamount of MTF-1 is located in the nucleus in the non-inducedstate, as assayed byWestern5 (57) and EMSA (24) analyses, andin vivo footprinting studies show a detectable footprint atMREd in the absence of added metal (21), presumably due tothe binding of MTF-1. In addition, ChIP assays confirmed theinteraction of some MTF-1 with the MT-1 promoter in unin-duced cells (Fig. 9) and (19, 49). It is thus possible that MREd-bound MTF-1 allows NF1 to bind to one of the NF1 sites and,

with other general transcription factors, control basal tran-scription. Interestingly, the NF1a/Sp1a site is occupied both inthe basal and induced state (17, 21), and we showed by in vitroDNA binding competition experiments that the NF1a site hasan apparent higher affinity for NF1 that the NF1b site (data notshown). In metal-induced cells, MTF-1 would strongly bind toMREd and MREa, thus causing further changes in chromatinstructure and allowing NF1 to interact with the other NF1 siteand to induce transcription. Alternatively, binding of NF1 toone NF1 site is MTF-1-dependent, whereas binding to theother site is not.Gene promoter induction is often associated with histone

acetylation and recruitment of chromatin-remodeling com-plexes (58). It is likely that, following zinc-induced MTF-1DNA-binding, an ATP-dependent remodeling complex and ahistone acetyltransferase are recruited to the MT promoterleading to local changes in chromatin structure and the subse-quent binding of NF1, cofactors, andmediator proteins. In fact,MTF-1 DNA-binding activity is sensitive to histone modifica-tions as DNA binding and expression of MTF-1 increase inlymphosarcoma cells treated with inhibitors of histonedeacetylase (59). However, the ATPase chromatin-remodel-ing complex SWI/SNF does not appear to be required for theactivation of the MT promoter in response to metals (60).The identification of the putative chromatin-remodelingcomplexes and histone acetyltransferase recruited to theMTpromoter in response to metal induction and the elucidationof the mechanisms of action of these factors will be a chal-lenging task in the future and may reveal new insights onmetal-regulated transcription.

Acknowledgments—We thank Dr. Nicolas Mermod for providingthe NF1 expression vectors and Dr. Jacques Pouyssegur for thepNHE1-R-1 vector. We are grateful to Alan Anderson for criticalreading of the manuscript and to Jacques Cote and Amine Nouranifor helpful suggestion and discussion on chromatin structure andChIP experiments.

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Tremblay, Geneviève St-Gelais, Manjapra V. Govindan and Carl SéguinOlivier LaRochelle, Simon Labbé, Jean-François Harrisson, Carl Simard, Véronique

Mouse Metallothionein-1 Gene in Response to Metal IonsNuclear Factor-1 and Metal Transcription Factor-1 Synergistically Activate the

doi: 10.1074/jbc.M800640200 originally published online January 29, 20082008, 283:8190-8201.J. Biol. Chem.

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