Sequence-Specific DNA Binding by the {alpha}NAC Coactivator Is Required for Potentiation of c-Jun-Dependent Transcription of the Osteocalcin Gene

Since the c-Jun coactivator ${alpha}$ NAC was initially identified ina differential screen for genes expressed in differentiatedosteoblasts, we examined whether the osteocalcin gene, a specificmarker of terminal osteoblastic differentiation, could be anatural target for the coactivating function of ${alpha}$ NAC. We hadalso previously shown that ${alpha}$ NAC can specifically bind DNA invitro, but it remained unclear whether the DNA-binding functionof ${alpha}$ NAC is expressed in vivo or if it is required for coactivation.We have identified an ${alpha}$ NAC binding site within the murine osteocalcingene proximal promoter region and demonstrated that recombinant ${alpha}$ NAC or ${alpha}$ NAC from ROS17/2.8 nuclear extracts can specificallybind this element. Using transient transfection assays, we haveshown that ${alpha}$ NAC specifically potentiated the c-Jun-dependenttranscription of the osteocalcin promoter and that this activityspecifically required the DNA-binding domain of ${alpha}$ NAC. Chromatinimmunoprecipitation confirmed that ${alpha}$ NAC occupies its bindingsite on the osteocalcin promoter in living osteoblastic cellsexpressing osteocalcin. Inhibition of the expression of endogenous ${alpha}$ NAC in osteoblastic cells by use of RNA interference provokeda decrease in osteocalcin gene transcription. Our results showthat the osteocalcin gene is a target for the ${alpha}$ NAC coactivatingfunction, and we propose that ${alpha}$ NAC is specifically targeted tothe osteocalcin promoter through its DNA-binding activity asa means to achieve increased specificity in gene transcription.

INTRODUCTION

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Introduction

The calcium-binding osteocalcin protein is a terminal differentiationmarker of the osteoblastic lineage. Dissection of the regulatorysequences controlling the osteoblast-specific expression ofthe osteocalcin gene has significantly improved our understandingof the molecular mechanisms regulating transcription duringskeletogenesis, for example, through identification of Runx2/Cbfa1as an osteoblast differentiation factor (22, 25). Characterizationof osteocalcin gene transcription has uncovered several regulatoryelements in the osteocalcin promoter (26), including bindingsites for the AP-1 family of transcription factors (2, 5, 16,28, 33-35, 40, 41).

The AP-1 family member c-Jun interacts with coactivators topotentiate transcription. One such coactivator has been characterizedas ${alpha}$ NAC (30, 36-38). The ${alpha}$ NAC protein, first identified as a regulatorof protein translation (45), was subsequently shown to alsofunction as a transcriptional coactivator by potentiating theactivities of the chimeric Gal4-VP16 activator (47) and of c-Junhomodimers (30, 36-38). ${alpha}$ NAC provides a protein bridge betweenc-Jun and the basal transcriptional machinery by contactingthe general transcription factor TBP (47). This stabilizes thec-Jun dimers on their cognate response element and results inenhanced transcription rates (30). In the course of those studies,it was shown that ${alpha}$ NAC can specifically bind DNA, although itdoes not act as a transcription factor (48). It remained unclearwhether the DNA-binding function of ${alpha}$ NAC is expressed or if itis masked in vivo and whether it is required for the coactivatingactivity of ${alpha}$ NAC.

The targeting of coactivators to particular promoters throughsequence-specific DNA binding is a means to achieve increasedspecificity in gene transcription. For example, the B-cell specificityof octamer promoters is due to the interaction of the ubiquitousOct-1 or lymphoid-specific Oct-2 factors with the B-cell-specificcoactivator Bob-1 (24). To prevent the widespread activationof any promoter recognized by Oct-1 or Oct-2, Bob-1 activityis restricted to particular sites by virtue of its sequence-specificDNA-binding activity (9, 18). Similarly, certain TAF coactivatorshave been shown to bind core promoter elements, including theinitiator region and the downstream promoter element (17).

Considering that we cloned ${alpha}$ NAC as a differentially expressedprotein in differentiated osteoblasts (30, 47), we examinedwhether the osteocalcin gene, a specific marker of terminalosteoblastic differentiation, could be a natural target forthe coactivating activity of ${alpha}$ NAC. We have identified an ${alpha}$ NACbinding site within the murine osteocalcin gene proximal promoterregion and demonstrated that recombinant ${alpha}$ NAC or ${alpha}$ NAC from nuclearextracts of osteoblastic ROS17/2.8 cells can specifically bindthis element. Using transient transfection assays, we have shownthat ${alpha}$ NAC potentiated the c-Jun-dependent transcription of theosteocalcin promoter and that this activity specifically requiredthe DNA-binding domain of ${alpha}$ NAC or the intact ${alpha}$ NAC binding sitewithin the promoter region. Using chromatin immunoprecipitation,we observed that ${alpha}$ NAC occupied its cognate binding site on theosteocalcin promoter in differentiated osteoblastic cells thatexpress osteocalcin. Finally, inhibition of endogenous ${alpha}$ NAC expressionby use of siRNAs affected osteocalcin promoter activity in osteoblasticcells. Our results show that the osteocalcin gene is a naturaltarget for the ${alpha}$ NAC coactivating function and that ${alpha}$ NAC is specificallytargeted to the osteocalcin promoter, presumably as a meansto achieve increased specificity in gene transcription.

MATERIALS AND METHODS

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Materials and Methods

Abbreviations. ${alpha}$ NAC, nascent polypeptide associated complex and coactivatoralpha; Runx2/Cbfa1, Runt family member x2/Core-binding factoralpha 1; AP-1, activating protein 1; Oct, octamer-binding protein;Bob-1, B-cell Oct-binding protein 1; OCN, osteocalcin; mmp-9,matrix metalloproteinase 9; OC box 1, osteocalcin box 1; ChIP,chromatin immunoprecipitation; DBD, DNA-binding domain; siRNA,small interfering RNA; TBP, TATA-binding protein; TAF, TBP-associatedfactors; EMSA, electrophoretic mobility shift assay; TBE, Tris-borate-EDTA;SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis.

Expression vectors and reporter plasmids. The p1316OG2-Luc reporter construct (herein referred to as OCN-Luc)(13) and the pCMV-Osf2 expression vector (14) (for expressionof full-length Runx2/Cbfa1) were generous gifts from GérardKarsenty (Baylor College of Medicine, Houston, TX). The mmp-9pGL3 reporter (MMP9-Luc), which contains the proximal 670 bpof the mmp-9 gene promoter driving luciferase (19), was providedby Shoukat Dedhar (University of British Columbia, Vancouver,British Columbia, Canada). To engineer the OCN-Luc ${Delta}$ BDG reporter,p1316OG2-Luc was digested at a unique Bst-AP1 restriction sitewithin the osteocalcin promoter. The plasmid was then digestedwith Bal-31 nuclease following the manufacturer's procedure(New England Biolabs, Pickering, Ontario, Canada). The Bal-31-digestedblunt-ended DNA fragments were self-ligated and used to transformEscherichia coli. Clones in which the ${alpha}$ NAC binding site (positions–35 to –45 relative to the transcription start site)was deleted (plasmid OCN-Luc ${Delta}$ BDG; the deletion covered from–32 to –60) were confirmed by direct DNA sequencing.Site-specific mutagenesis of the ${alpha}$ NAC binding site within themurine osteocalcin promoter fragment (GCACgGgGTAG [lowercaseindicates mutated residues]; OCN-Luc mut BDG reporter) was performedby NorClone Biotech Laboratories (London, Ontario, Canada) usinginverse PCR. The expression vectors for the Flag epitope-taggedfull-length ${alpha}$ NAC (pSI-NAC-Flag) (36) and for full-length c-Jun(pCI-cJun) (38) have been described previously. The cDNA forthe ${Delta}$ 69-80 deletion mutant ( ${alpha}$ NAC ${Delta}$ 69-80) was obtained by PCR cloningand inserted in-frame into pSI-Flag (36) to yield the pSI- ${alpha}$ NAC ${Delta}$ 69-80-Flagexpression vector.

Transfections. COS-7 African green monkey cells were transfected using Lipofectaminereagent as described previously (36). Transfections utilized300 ng of pCi-cJun alone or in combination with 300 ng of pSI-NAC-Flagor pSI- ${alpha}$ NAC ${Delta}$ 69-80-Flag. In one series of transfections, pCi-cJunwas replaced by pCMV-Osf2. One hundred nanograms of reporterplasmids was used, and 40 ng of pSV6tk-CAT (11) was added toeach sample as a control of transfection efficiency. All transfectionswere conducted in triplicate, and values are reported as means± standard errors of the means. Statistical analysiswas performed using analysis of variance and the Tukey or Bonferroniposttests. P values of <0.05 were accepted as significant.The expression level of transfected proteins was controlledby immunoblotting following immunoprecipitation with anti-Flagor anti-c-Jun antibodies (38).

EMSA. Complementary oligonucleotides corresponding to the putative ${alpha}$ NAC binding site within the murine osteocalcin proximal promoterregion (5'-GAGAGCACAGAGTAGCCGA-3', positions –49 to –32relative to the transcription start site [the putative bindingsite is shown in italics]) were synthesized with an overhang,annealed, and labeled with ³²P-labeled deoxynucleoside triphosphatesby Klenow fill-in using standard protocols (3). One probe (seeFig. 2C) covered the ${alpha}$ NAC binding site and the OC box 1 of theosteocalcin promoter, positions –103 to –30 relativeto the transcription start site. Competitions used unlabeleddouble-stranded oligonucleotides corresponding to the previouslyidentified ${alpha}$ NAC binding site (5'-GAGACGACACACAGGCCGA-3'; 1x NAC)(48) or the mutated osteocalcin sequence (5'-GAGAGCACgGgGTAGCCGA-3';mut).

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FIG. 2. ${alpha}$ NAC and c-Jun bind the osteocalcin proximal promoter. EMSAs using a probe from the murine osteocalcin gene and purified recombinant ${alpha}$ NAC (A), nuclear extracts (NE) from ROS 17/2.8 cells (B), or recombinant c-Jun and recombinant ${alpha}$ NAC (C). In panel A, a specific complex can be detected ( ${alpha}$ NAC arrow) that was "supershifted" in the presence of anti- ${alpha}$ NAC antibodies (Ab). Preimmune serum (Serum) did not influence complex migration. The complex was competed with increasing amounts of the canonical ${alpha}$ NAC binding site (1xNAC) but not with a mutated sequence (mut). DBD-deleted ${alpha}$ NAC ( ${Delta}$ 69-80) did not bind the probe (lane 10). Panel B shows that ${alpha}$ NAC from ROS 17/2.8 osteoblastic cells bound the probe. The complex was specifically but not completely supershifted by the anti- ${alpha}$ NAC antibody (Ab). In panel C, the probe used covered positions –103 to –30 of the osteocalcin promoter and encompassed the ${alpha}$ NAC binding site and the OC box 1 (34). Recombinant c-Jun did not bind the probe by itself (lane 2), but a complex containing c-Jun could be detected (c-Jun arrow) when the probe was incubated with both c-Jun and ${alpha}$ NAC (lanes 4 and 5).

The recombinant ${alpha}$ NAC proteins (wild-type and ${Delta}$ 69-80) were purifiedusing NEB's IMPACT system as described previously (38). ROS17/2.8nuclear extracts were prepared following the technique of Dignamet al. (12) as detailed elsewhere (7). Purified, recombinantc-Jun protein was purchased from Promega (Madison, WI).

Recombinant proteins or nuclear extracts were incubated for30 min at 4°C in 20 µl of binding buffer [100 mM HEPES,pH 7.5, 20 mM MgCl₂, 500 mM NaCl, 2% NP-40, 10 mM dithiothreitol,10 mM EDTA, 100 ng of poly(dI-dC), 30% glycerol]. Labeled probe(5,000 dpm) was added to the binding reaction mixture. For competitionexperiments, the binding reaction was incubated with excessunlabeled 1x NAC or mut oligonucleotides. For supershift assays,anti- ${alpha}$ NAC antibody (48) or anti-c-Jun antibody (Santa Cruz BiotechnologyInc., Santa Cruz, CA) was added to the binding reaction for15 min prior to the addition of the labeled probe. The boundmixtures were size fractionated on a nondenaturing 6% polyacrylamidegel at 140 V for 4 h in 0.5x TBE buffer. The gels were subsequentlydried and autoradiographed.

ChIP. MC3T3-E1 cells (44) were plated on 100-mm-diameter dishes for14 or 21 days in mineralizing media (10 mM ß-glycerophosphateand 50 µg/ml ascorbic acid). Cells were then cross-linkedby adding formaldehyde (1%, 20 min) with gentle agitation. Thecross-linking was stopped by the addition of glycine to a finalconcentration of 125 mM for 10 min. The cells were then washedthree times with ice-cold phosphate-buffered saline, scraped,and collected by centrifugation in 1 ml phosphate-buffered salineplus protease inhibitors (1 µg/ml leupeptin, 1 µg/mlaprotinin, and 1 mM phenylmethylsulfonyl fluoride). The cellpellet was resuspended for 10 min on ice in lysis buffer {5mM PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid)], pH 8.0,85 mM KCl, and 0.5% NP-40, with protease inhibitors as describedabove}. The nuclei were pelleted by centrifugation (5,000 rpm,5 min), resuspended in 1.5 ml of nuclear lysis buffer (50 mMTris-HCl, pH 8.1, 10 mM EDTA, 1% SDS, and protease inhibitors),and incubated on ice for 10 min.

To reduce the length of the chromatin fragments to approximately600 bp, the extract was sonicated with a Sonic Dismembrator(model 500, Fisher Scientific Ltd., Nepean, Ontario, Canada),using four 15-s pulses at 30% of maximal power. After centrifugation(12,000 x g, 10 min, 4°C), the supernatant containing thechromatin was collected. Cross-linked extracts were dilutedfivefold in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100,1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl, proteaseinhibitors). Immunoprecipitations were performed with Sepharose-conjugatedanti-Flag monoclonal antibody (Sigma-Aldrich, St. Louis, MO),anti- ${alpha}$ NAC polyclonal antibody (48), anti-IgG-agarose (as a negativecontrol), or anti-Runx2/Cbfa1 polyclonal antibody (Santa Cruz).The immune complexes (anti- ${alpha}$ NAC and anti-Runx2) were recoveredwith the addition of 150 µl of protein A-Sepharose beadsand a subsequent incubation for 3 h at 4°C with agitation.The complexes were washed for 5 min with 1 ml of each of thefollowing solutions: low-salt wash buffer (0.1% SDS, 1% TritonX-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), high-saltwash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl,pH 8.1, 500 mM NaCl), and LiCl wash buffer (250 mM LiCl, 1%NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.0).Finally, they were washed twice in 1x Tris-EDTA buffer (10 mMTris-HCl, pH 8.0, 1 mM EDTA).

The protein-DNA complexes were eluted by two incubations with250 µl of elution buffer (100 mM NaHCO₃, 1% SDS) withagitation and then centrifuged (10,000 x g for 3 min). To reversecross-linking, the supernatant was collected and incubated with1 µl of 10-mg/ml RNaseA in 300 mM NaCl at 65°C for4 h. Then, the proteins were digested with 200 µg of proteinaseK per ml for 1 h at 45°C. The DNA was precipitated overnightwith ethanol at –20°C. DNA fragments were purifiedwith a QIAquick Spin instrument (QIAGEN Inc.-Canada, Mississauga,Ontario, Canada). The sequences of the primers used were asfollows. For ${alpha}$ NAC forward, 5'-AGGCAGCTGCAATCACCA-3' (beginningat nucleotide –153 of the murine osteocalcin promotersequence) was used, and for ${alpha}$ NAC reverse, 5'-GCACCCTGCAGCATCCA-3'(position –2) was used. For Runx2/Cbfa1, the primers usedwere as published by Shen et al. (42): forward, 5'-AAATGAGGACATTACTGAACACTCC-3'(position –459); and reverse, 5'-CCAAAGGATGCTGTGGTTGGTGAT-3'(position –118).

Northern blot assay. Total RNA was isolated with the Trizol reagent (Life TechnologiesInc., Burlinton, Ontario, Canada), and Northern blots were performedusing a standard methodology. The probe used was the 470-bpEcoRI-PstI fragment from the mouse osteocalcin cDNA (8).

RNA interference. A subclone of MC3T3-E1 preosteoblasts showing high differentiation/mineralizationpotential and stably transfected with 1.3 kb of the mouse osteocalcingene promoter driving expression of luciferase (46), a generousgift of Renny T. Franceschi (University of Michigan), was culturedat confluence for 7 days in the presence of G418. The cellswere then passaged onto gelatin-coated wells at 20,000 cells/wellin 24-well plates. The following day, the cells were transfectedwith 100 nM of siRNA directed against ${alpha}$ NAC (catalog number 156855;Ambion Inc., Austin, TX) using 2.5 µl of siPORT Amine(Ambion) according to the manufacturer's instructions. Controlwells were treated with the siPORT reagent only or were transfectedwith siRNA control no. 1, a sequence that shows no homologyto any inventoried gene in the mouse or human databases (Ambion).The transfection mixture was left on the cells for 8 h, andthen 1 ml of complete medium was added. RNA was harvested at32 h posttransfection using the RNAqueous-4PCR kit (Ambion).The RNA was reverse-transcribed into cDNA using a High-CapacitycDNA Archive Kit per the manufacturer's recommendations (AppliedBiosystems, Foster City, CA). Real-time PCR amplification wasperformed on an Applied Biosystems 7700 instrument using theTaqMan Universal PCR Master Mix (Applied Biosystems) and specificTaqMan assays for ${alpha}$ NAC, OCN, and GAPDH. The expression levelof each mRNA was quantified by the ${Delta}$ ${Delta}$ C_T method (User bulletinno. 2, ABI Prism 7700 sequence detection system) and normalizedto GAPDH levels. For luciferase activity, cell lysates wereprepared in the reporter gene assay lysis buffer (Roche MolecularBiochemicals, Laval, Quebec, Canada). One hundred microlitersof cell lysate was used for single luciferase reporter assaysfollowing the manufacturer's instructions (Promega). Luciferaseactivity was measured with a Sirius luminometer (Berthold DetectionSystems GmbH, Pforzheim, Germany). The expression level detectedin cells treated with the transfection reagent alone was arbitrarilygiven a value of 1. Statistical significance was determinedby analysis with an unpaired, one-tailed Student's t test fortwo-group comparison or by analysis of variance followed bythe Dunnett's posttest for multiple group comparison. P valuesof <0.05 were accepted as significant.

RESULTS

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Results

${alpha}$ NAC specifically potentiates c-Jun-dependent transcription from the mouse osteocalcin promoter. Since we identified the c-Jun coactivator ${alpha}$ NAC as a differentiallyexpressed gene product in terminally differentiated mouse osteoblasts(30, 47), we tested whether the osteocalcin gene, a marker ofthe mature osteoblastic phenotype and an AP-1 target (2, 5,16, 28, 33-35, 40, 41), would respond to the coactivating activityof ${alpha}$ NAC. A reporter construct in which the luciferase gene wasplaced downstream from the murine osteocalcin gene promoter(13) was cotransfected with expression vectors for c-Jun and/or ${alpha}$ NAC. Figure 1 shows that c-Jun activated transcription fromthe mouse osteocalcin gene promoter (bar 2) and that this effectwas potentiated by cotransfection with full-length, wild-type ${alpha}$ NAC (bar 4). Since it is a coactivator, not a transcriptionfactor, expression of ${alpha}$ NAC by itself did not influence reportergene expression (bar 3), as previously observed with other promoters(36, 48). The coactivating activity of ${alpha}$ NAC was specific to c-Jun,as cotransfecting ${alpha}$ NAC with Runx2/Cbfa1, a key regulator of osteocalcingene transcription (14, 22, 27), had no effect on Runx2/Cbfa1-mediatedtranscription (Fig. 1, bars 5 and 6). The expression of thetransfected FLAG-NAC fusion protein was monitored by immunoblottingusing the anti- ${alpha}$ NAC antibody (48), which also detects the endogenousprotein (Fig. 1B). Since each transfection was performed atleast three times in triplicate with identical results, we areconfident that the lack of coactivation activity of ${alpha}$ NAC withRunx2/Cbfa1 was not due to the somewhat lower expression levelsobserved in the immunoblot shown in Fig. 1B. We conclude that ${alpha}$ NAC specifically potentiates the c-Jun-mediated transcriptionof the osteocalcin gene.

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FIG. 1. ${alpha}$ NAC specifically potentiates the transcription of the osteocalcin gene promoter by c-Jun. (A) COS-7 cells were transiently transfected with an osteocalcin-luciferase reporter and expression vectors for c-Jun, ${alpha}$ NAC, or Runx2/Cbfa1, alone or in combinations. c-Jun induced the transcription of the reporter, and this induction was potentiated by ${alpha}$ NAC. The coactivating function of ${alpha}$ NAC was specific to c-Jun, as ${alpha}$ NAC expression did not affect Runx2/Cbfa1-activated osteocalcin gene transcription. The expression level detected in cells transfected with the reporter construct alone was arbitrarily given a value of 1. Results are means ± standard errors of the means of three independent transfections performed in triplicate. *, P < 0.05; ***, P < 0.001. (B) Immunoblot probed with the anti- ${alpha}$ NAC antibody that shows expression levels of endogenous and FLAG-tagged ${alpha}$ NAC. The order of the numbered tracks corresponds to the order of the bars in panel A.

${alpha}$ NAC specifically binds the osteocalcin proximal promoter region. We have previously reported that ${alpha}$ NAC can bind the degeneratesequence 5'-C/G C/G A C/G A C/G A nnn G-3', where n is any nucleotide(48). We identified a matching sequence between positions –35and –45 of the mouse osteocalcin promoter (5'-GCACAGAgtaG-3')and used EMSA to determine whether ${alpha}$ NAC could specifically bindthis element. A labeled, double-stranded probe correspondingto positions –49 to –32 relative to the osteocalcintranscription start site was synthesized and used in EMSA withpurified recombinant ${alpha}$ NAC. Figure 2A shows that the recombinantprotein formed a strong complex with the labeled probe (lane3) that was supershifted in the presence of a specific anti- ${alpha}$ NACantibody (lane 4) (48). A less intense complex with slower electrophoreticmobility that was not as affected by the antibody was also observed.This complex could be due to differentially phosphorylated formsof ${alpha}$ NAC (36, 37). Preimmune serum or an excess of unlabeled mutatedoligonucleotide did not influence binding (lanes 5, 6), whileincreasing amounts of an unlabeled oligonucleotide correspondingto the previously characterized ${alpha}$ NAC binding site (48) efficientlycompeted binding of both complexes (lanes 7 to 9). An unrelatedprotein (maltose-binding protein) purified using the same systemthat was used for the purification of recombinant ${alpha}$ NAC proteinsdid not bind the probe (lane 2), confirming that the bindingactivity was not due to a contaminant from the protein purificationsystem. Nuclear extracts from the osteoblastic cell line ROS17/2.8 also formed a complex with the labeled probe that wasspecifically but not completely supershifted by the anti- ${alpha}$ NACantibody, demonstrating that nuclear ${alpha}$ NAC from osteoblasts couldbind the osteocalcin sequence (Fig. 2B, lanes 2 to 4).

We have engineered a series of C-terminal and internal deletionmutants of the ${alpha}$ NAC protein (37, 38) and tested them for DNA-bindingactivity with EMSA (data not shown). We found that deletingresidues 69 to 80 of the 215-amino-acid ${alpha}$ NAC protein abrogatedthe ability of the mutated protein ( ${alpha}$ NAC ${Delta}$ 69-80) to bind DNA (Fig.2A, lane 10). These experiments mapped the ${alpha}$ NAC DNA-binding domainto residues 69 to 80 and showed that recombinant ${alpha}$ NAC, as wellas ${alpha}$ NAC from osteoblastic nuclear extracts, could specificallybind the osteocalcin proximal promoter region in EMSA.

We also tested the binding of recombinant c-Jun to the mouseosteocalcin promoter. We used an oligonucleotide probe coveringresidues –103 to –30 of the mouse promoter. Thisprobe included the ${alpha}$ NAC binding site (–45 to –35)and a sequence that is 100% homologous to the OC box 1 fromthe rat osteocalcin gene promoter (–99 to –76) (13,34). The rat OC box 1 was shown to bind Fos/Jun AP-1 complexes(34). As previously reported (34), c-Jun homodimers did notbind this element (Fig. 2C, lane 2). The probe efficiently boundrecombinant ${alpha}$ NAC (lane 3). Interestingly, when the probe wasincubated with both ${alpha}$ NAC and c-Jun, a novel complex formed (lane4). This complex contained c-Jun, since it was supershiftedby an anti-c-Jun antibody (lane 5). We interpret this resultto mean that ${alpha}$ NAC can stabilize the binding of c-Jun homodimersto the osteocalcin promoter, as was previously described forthe AP-1 site from the metallothionein-IIA promoter (30).

${alpha}$ NAC must bind the osteocalcin promoter to coactivate c-Jun-dependent osteocalcin gene transcription. Transient transfection assays were used to determine the importanceof the binding of ${alpha}$ NAC to the osteocalcin promoter for its coactivatingactivity. In the first series of experiments, the activity ofthe DBD-deleted mutant ( ${alpha}$ NAC ${Delta}$ 69-80) was compared to the activityof intact ${alpha}$ NAC. Figure 3A shows that contrary to its wild-typecounterpart, ${alpha}$ NAC ${Delta}$ 69-80 could not potentiate c-Jun-mediated osteocalcingene transcription. Both ${alpha}$ NAC and ${alpha}$ NAC ${Delta}$ 69-80 were efficiently expressedin those experiments (Fig. 3B, second panel). The FLAG-tagged ${alpha}$ NAC proteins were expressed at levels similar to that of endogenous ${alpha}$ NAC (Fig. 3B, third panel) (note that FLAG- ${alpha}$ NAC ${Delta}$ 69-80 comigrateswith endogenous ${alpha}$ NAC, and thus the anti- ${alpha}$ NAC antibody recognizesboth proteins at the same position in the gel).

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FIG. 3. Coactivation of c-Jun-dependent osteocalcin gene transcription requires the ${alpha}$ NAC DBD. (A) Transient transfection assays were set up as described in the legend to Fig. 1. The recombinant ${alpha}$ NAC ${Delta}$ 69-80 protein, devoid of DNA-binding activity, did not potentiate the activity of c-Jun. **, P < 0.01; ***, P < 0.001. (B) The expression of the recombinant proteins was monitored by Western blot assay using the antibodies (Ab) listed above each panel. Probing with anti-TBP served as a loading control. Note that FLAG- ${alpha}$ NAC ${Delta}$ 69-80 migrates at the same position as endogenous ${alpha}$ NAC in SDS-PAGE, so that the band detected by the anti- ${alpha}$ NAC antibody (third panel, lanes 5 and 6) represents the combined signal of endogenous ${alpha}$ NAC and FLAG- ${alpha}$ NAC ${Delta}$ 69-80.

The ${alpha}$ NAC binding element was then mutated or deleted from theosteocalcin promoter. While c-Jun could still increase transcriptionfrom both mutated reporters (OCN-Luc ${Delta}$ BDG and OCN-Luc mut BDG;Fig. 4A and B, respectively), wild-type ${alpha}$ NAC could not potentiatec-Jun-dependent expression of the reporter gene when its bindingsite was mutated (Fig. 4B, bar 8) or removed from the osteocalcinpromoter (Fig. 4A, bar 6). Taken together, those data show that ${alpha}$ NAC must interact with the promoter to coactivate osteocalcingene expression induced by c-Jun.

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FIG. 4. Coactivation of c-Jun-dependent osteocalcin gene transcription requires the ${alpha}$ NAC binding site on the promoter. Transient transfection assays were performed as described in the legend to Fig. 1. (A) Reporters used included the wild-type osteocalcin promoter driving luciferase (OCN-Luc) or a mutated osteocalcin promoter in which the ${alpha}$ NAC binding site was deleted (OCN-Luc ${Delta}$ BDG; the deletion covered from –32 to –60 relative to the transcription start site). (B) Reporters used included OCN-Luc and a mutated osteocalcin promoter in which the ${alpha}$ NAC binding site was mutated by site-specific mutagenesis (OCN-Luc mut BDG; the engineered mutation was 5'-GCACgGgGTAG-3'). ${alpha}$ NAC did not potentiate the activity of c-Jun when its binding site was mutated or deleted from the promoter. **, P < 0.01; ***, P < 0.001.

The ${alpha}$ NAC DNA-binding domain is specifically required for osteocalcin expression. To determine whether ${alpha}$ NAC must always bind DNA to exert its coactivatingactivity, we compared the activity of wild-type ${alpha}$ NAC and theDBD mutant, ${alpha}$ NAC ${Delta}$ 69-80, on two natural target promoters, osteocalcinand mmp-9 (36). As described above, ${alpha}$ NAC ${Delta}$ 69-80 could not potentiatec-Jun-mediated transcription from the OCN-Luc template (Fig.5). Transcription from the mmp-9 promoter is induced by c-Jun,and wild-type ${alpha}$ NAC potentiates this activity (Fig. 5, bars 7to 10) (36). Transfection of ${alpha}$ NAC ${Delta}$ 69-80 by itself led to an increasein MMP9-Luc expression. Interestingly, the DBD-deleted mutantof ${alpha}$ NAC potently coactivated c-Jun-dependent transcription fromthe mmp-9 promoter (Fig. 5, bar 12). We interpret these datato mean that the ${alpha}$ NAC DNA-binding activity is not always necessaryfor coactivation, but that it is a specific requirement forthe potentiation of c-Jun-dependent transcription from the osteocalcinpromoter.

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FIG. 5. ${alpha}$ NAC DNA-binding activity is specifically required for c-Jun-dependent osteocalcin gene transcription. Transient transfection assays were set up as described in the legend to Fig. 1. Two c-Jun responsive promoters were used as reporter constructs: the wild-type osteocalcin promoter (OCN-Luc) or the proximal 670 bp of the mmp-9 gene promoter (MMP9-Luc). Wild-type ${alpha}$ NAC could potentiate the activity of c-Jun on both promoters, but the DNA-binding domain-deleted ${alpha}$ NAC mutant ( ${alpha}$ NAC ${Delta}$ 69-80) was active only on the MMP9-Luc template. **, P < 0.01; ***, P < 0.001.

The ${alpha}$ NAC-DNA interaction occurs in vivo. We used chromatin immunoprecipitation to determine if ${alpha}$ NAC occupiesits cognate binding site on the osteocalcin promoter in livingcells expressing osteocalcin. Wild-type osteoblastic MC3T3-E1cells or MC3T3-E1 cells stably transfected with pSI-NAC-Flagor FLAG- ${alpha}$ NAC ${Delta}$ 69-80 were grown for 14 or 21 days postconfluencein the presence of ascorbic acid and beta-glycerophosphate.The expression of the wild-type or fusion proteins was monitoredby immunoblotting (Fig. 6B). Note that FLAG- ${alpha}$ NAC ${Delta}$ 69-80 migratesat the same position as endogenous ${alpha}$ NAC in SDS-PAGE, so thatthe band detected by the anti- ${alpha}$ NAC antibody, shown in Fig. 6B,lane 3, represents the combined signal of endogenous ${alpha}$ NAC andFLAG- ${alpha}$ NAC ${Delta}$ 69-80. Osteocalcin gene expression can readily be observedafter 14 days in culture under the conditions used (15) (Fig.6C). DNA was cross-linked to proteins using formaldehyde, andsonicated chromatin from whole-cell lysates was coimmunoprecipitatedusing specific or negative control antibodies. DNA fragmentsthat coprecipitated with the target proteins were purified uponreversal of protein/DNA cross-links and used as templates forPCRs with osteocalcin-specific primers. As a positive controlfor the assay, we used antibodies directed against Runx2/Cbfa1and previously published primer pairs (42). The 350-bp amplimerdiagnostic of Runx2/Cbfa1 binding to the osteocalcin promoter(42) was readily observed (Fig. 6A and D, lanes 1 and 2).

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FIG. 6. ${alpha}$ NAC binds the osteocalcin gene promoter in living cells expressing osteocalcin. Wild-type osteoblastic MC3T3-E1 cells or MC3T3-E1 cells stably transfected with pSI-NAC-Flag (A) or the Flag epitope-tagged DBD-deleted ${alpha}$ NAC mutant, ${alpha}$ NAC ${Delta}$ 69-80 (D), were grown for 14 (panel A) or 21 (panels A and D) days postconfluence in the presence of ascorbic acid and beta-glycerophosphate. Immunoprecipitation assays were performed with formaldehyde-cross-linked chromatin and antibodies against Runx2/Cbfa1, ${alpha}$ NAC, or the Flag epitope. Ethidium bromide-stained agarose gels of PCR products obtained with primers flanking the Runx2/Cbfa1 binding site (lanes 1 and 2) or the ${alpha}$ NAC binding site (lanes 3 to 9) within the mouse osteocalcin gene promoter are shown. Input, amplification of DNA prior to immunoprecipitation; IgG, immunoglobulin G; I.P., immunoprecipitate; M, molecular size markers. (B) Immunoblot probed with the anti- ${alpha}$ NAC antibody that shows expression levels of endogenous and FLAG-tagged ${alpha}$ NAC proteins. Note that FLAG- ${alpha}$ NAC ${Delta}$ 69-80 migrates at the same position as endogenous ${alpha}$ NAC in SDS-PAGE, so that the band detected by the anti- ${alpha}$ NAC antibody (lane 3) represents the combined signal of endogenous ${alpha}$ NAC and FLAG- ${alpha}$ NAC ${Delta}$ 69-80. (C) Northern blot showing OCN mRNA expression. 28S, ribosomal 28S RNA used to monitor loading.

Cross-linked DNA was then immunoprecipitated using anti- ${alpha}$ NACor anti-Flag antibodies and amplified using primer pairs thatflank the ${alpha}$ NAC binding site within the osteocalcin proximal promoter.The diagnostic 151-bp amplimer could be observed using bothantibodies (Fig. 6A, lanes 5 to 8), and the amount of amplifiedDNA correlated with osteocalcin gene expression (Fig. 6A and C).No DNA was amplified when nonspecific IgGs were used forimmunoprecipitation (Fig. 6A, lane 4). Similarly, the anti-Flagantibody did not immunoprecipitate DNA from untransfected MC3T3-E1cells at 21 days postconfluence (lane 9). As an additional negativecontrol, primer pairs selected from the osteocalcin coding sequencewere selected. These primers did not amplify chromatin immunoprecipitatedwith either the anti- ${alpha}$ NAC or anti-Flag antibodies (data not shown).

MC3T3-E1 cells were also stably transfected with the Flag epitope-taggedDBD-deleted ${alpha}$ NAC mutant, ${alpha}$ NAC ${Delta}$ 69-80 (Fig. 6B, lane 3). In thosetransfectants, an osteocalcin promoter fragment was coimmunoprecipitatedwith the endogenous wild-type ${alpha}$ NAC (Fig. 6D, lane 3), while theanti-Flag antibody, which precipitated ${alpha}$ NAC ${Delta}$ 69-80 (not shown),did not coimmunoprecipitate osteocalcin chromatin (Fig. 6D,lane 4).

These data show that in living osteoblastic cells expressingosteocalcin, ${alpha}$ NAC occupies its cognate binding element. Theyfurther confirm that the ${alpha}$ NAC ${Delta}$ 69-80 deletion mutant is devoidof DNA-binding activity.

Inhibition of endogenous ${alpha}$ NAC expression affects osteocalcin promoter activity. To confirm the relevance of ${alpha}$ NAC in the control of osteocalcingene transcription, we inhibited endogenous ${alpha}$ NAC expression usingRNA interference. These experiments were performed using a subcloneof MC3T3-E1 preosteoblasts exhibiting high differentiation/mineralizationpotential that has been stably transfected with a luciferasereporter gene under the control of the 1.3-kb mouse osteocalcinpromoter fragment (46). The transcriptional control of thisstably integrated reporter allele was shown to be identicalto that of the endogenous gene (46). Specific siRNAs for ${alpha}$ NACinhibited 90% of the ${alpha}$ NAC mRNA when quantified by real-time reversetranscription-PCR (Fig. 7A) and considerably reduced ${alpha}$ NAC proteinexpression over the duration of the experiment (Fig. 7B). Theefficiency of the ${alpha}$ NAC siRNA in inhibiting its target was comparedto that of an unrelated control siRNA (Fig. 7A and B, lane 2)or to the transfection reagent alone (Fig. 7B, lane 1). We nextassessed the impact of ${alpha}$ NAC siRNA knock-down on osteocalcin genetranscription. Treatment of cells with the control siRNA affectedosteocalcin expression, which was lower in transfected cellsthan in untransfected controls (relative expression was calculatedas 26%) (Fig. 7A and C). This could be due to the experimentalconditions, which included the subculturing of confluent cellsinto sparse cultures combined with treatment with the transfectionreagent. Nevertheless, treatment of cells with the specific ${alpha}$ NAC siRNA that inhibited ${alpha}$ NAC mRNA and protein expression furtherrepressed both endogenous osteocalcin mRNA levels (Fig. 7A)and the transcription of the osteocalcin promoter-controlledluciferase reporter (Fig. 7C). The effect of the ${alpha}$ NAC siRNA treatmenton endogenous osteocalcin expression nearly reached statisticalsignificance (P = 0.0512) (Fig. 7A), while its impact on thetranscription of the reporter allele was highly significant(Fig. 7C). These data establish the physiological relevanceof endogenous ${alpha}$ NAC as a regulator of osteocalcin gene transcriptionin osteoblastic cells.

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FIG. 7. Inhibition of endogenous ${alpha}$ NAC expression by RNA interference affects osteocalcin promoter activity. MC3T3-E1 cells stably transfected with a luciferase reporter gene under the control of the 1.3-kb mouse osteocalcin promoter fragment (46) were transfected with a control, unrelated siRNA, or an siRNA directed against ${alpha}$ NAC. (A) Expression of the endogenous ${alpha}$ NAC or osteocalcin mRNAs was monitored by real-time PCR. ***, P < 0.001. (B) Expression of the endogenous ${alpha}$ NAC protein was assessed by immunoblotting with the anti- ${alpha}$ NAC antibody. Lane 1, treatment with the transfection reagent alone; lane 2, transfection with the control siRNA; lane 3, transfection with the ${alpha}$ NAC siRNA. Staining of the membrane with Ponceau red (bottom panel) was used to monitor for even loading of each lane on the gel. (C) Expression of the reporter luciferase gene under the control of the osteocalcin promoter was monitored with a luminometer. The control bar represents expression measured in the presence of the transfection reagent alone. **, P < 0.01; Ctrl siRNA, control siRNA. Treatment of cells with the specific ${alpha}$ NAC siRNA repressed both endogenous osteocalcin mRNA levels and the transcription of the osteocalcin promoter-controlled luciferase reporter.

DISCUSSION

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Discussion

We have previously shown that ${alpha}$ NAC functions as a c-Jun coactivator(30, 36-38). The cloning of ${alpha}$ NAC as a differentially expressedgene product in terminally differentiated osteoblasts (47) andthe expression of the ${alpha}$ NAC protein in mineralizing osteoblastsat the ossification centers of developing embryos (30) suggestedthat ${alpha}$ NAC should be involved in some aspects of the regulationof gene transcription in differentiated bone-forming cells.We have now identified the osteocalcin gene as a natural targetgene for ${alpha}$ NAC's coactivating function. Osteocalcin encodes abone-specific protein induced in osteoblasts with the onsetof mineralization at late stages of differentiation (32). Interestingly,we have found that the regulation of osteocalcin gene transcriptionby ${alpha}$ NAC specifically required the previously identified DNA-bindingactivity of ${alpha}$ NAC (48), and we have mapped the ${alpha}$ NAC DNA-bindingdomain between amino acid residues 69 and 80. Confirming thein vitro assays, we showed that ${alpha}$ NAC binds the osteocalcin proximalpromoter region in differentiated osteoblastic cells expressingosteocalcin and that specific inhibition of ${alpha}$ NAC by RNA interferenceaffects osteocalcin promoter activity.

Functional AP-1 binding sites have been characterized for thehuman (16, 35, 41) and rat (2, 5, 28, 33, 34) osteocalcin promoters.Transcriptional induction of the murine osteocalcin promoterby AP-1 family members has also been reported (40). The precisemouse osteocalcin AP-1 response element remains to be characterized,but we speculated that regions from the mouse gene showing homologyto characterized AP-1 elements in the promoter of the osteocalcingene from other species could be functional sites. The mostlikely candidate sequence would correspond to the "OC box 1"between nucleotides –75 and –98, which is 100% homologousbetween rat and mouse. The rat "OC box 1" sequence was shownto bind AP-1 molecules (34). Interestingly, the ${alpha}$ NAC bindingsite that we have identified is adjacent to "OC box 1." Whilerecombinant c-Jun did not bind the OC box 1 by itself (Fig.2C) (34), a binding complex that contained c-Jun was detectedwhen the transcription factor and coactivator were incubatedtogether with the probe (Fig. 2C). We propose that the bindingof c-Jun to its response element on the osteocalcin promoterrequires ${alpha}$ NAC, which has been previously shown to stabilize thebinding of c-Jun homodimers on canonical AP-1 sites (30). Thismodel would explain why previous efforts to detect homodimericc-Jun binding to the osteocalcin promoter were unsuccessful,since they did not include ${alpha}$ NAC in the binding reaction. Furtherstudies are under way to precisely delineate the c-Jun bindingsite and define the molecular determinants of the c-Jun- ${alpha}$ NACinteraction on the mouse osteocalcin gene promoter.

In differentiated rat osteoblasts, Fra-2/JunD heterodimers wereidentified as the major AP-1 complexes (28, 29). Our resultsshow that the mouse osteocalcin gene promoter can respond toc-Jun. It remains to be determined whether the transfected c-Junprotein dimerized with endogenous Fos family members to activateosteocalcin gene transcription. Dimerization with c-Fos appearsunlikely, as we have previously shown that ${alpha}$ NAC cannot potentiatethe activity of the c-Fos/c-Jun heterodimer (30). Potentialc-Jun dimerization partners would include Fra-1 or ${Delta}$ FosB, whichhave been shown to play functional roles in osteoblasts in theregulation of bone mass accrual (21, 23, 39, 40, 43). We favorthe possibility that the c-Jun homodimer regulates osteocalcingene transcription and that this effect is maximized in thepresence of the c-Jun coactivator, ${alpha}$ NAC. This interpretationcould be formally tested using the recently described expressionvectors for single-chain tethered AP-1 dimers (4).

Our results confirm the previously reported specific DNA-bindingactivity of ${alpha}$ NAC (48). This contrasts with published work claimingthat the nucleic acid-binding activity of NAC subunits is nonspecific(6). These studies also utilized recombinant ${alpha}$ NAC purified frombacteria, although they were purified with a different fusionprotein system. We cannot find obvious reasons for the discrepanciesbetween the results obtained in each case. The binding elementthat we identified herein within the osteocalcin promoter regionmatches the consensus sequence characterized with the PCR-basedtechnique involving selection and amplification of the bindingsite (48). The ${alpha}$ NAC binding site in the osteocalcin promoteris fairly conserved among species (see the promoter sequencecomparisons in reference 13), but the functionality of the homologoussequences from the rat and human promoters remains to be tested.It will be interesting to determine whether other genes containsimilar sequence elements within their regulatory regions, althoughpreliminary data suggest that homologous sequences are rarewithin the available databases (not shown).

We observed that ${alpha}$ NAC's DNA-binding activity was required forosteocalcin gene coactivation but was dispensable for the potentiationof c-Jun-dependent transcription of the mmp-9 gene. This raisesthe interesting possibility that ${alpha}$ NAC may be targeted to theosteocalcin promoter to achieve an increased specificity forgene transcription in differentiated osteoblasts. Such a mechanismhas already been described for the B-cell-specific coactivatorBob-1 (9, 18).

Chromatin immunoprecipitation demonstrated that ${alpha}$ NAC occupiedits cognate binding site in intact osteoblasts expressing osteocalcin.In nonexpressing cells, several mechanisms could operate. Thecoactivator might be prevented from contacting its binding elementeither by the inaccessibility of the chromatin or by posttranslationalregulation. The first hypothesis is supported by the well-characterizedrole of chromatin-modifying complexes in the regulation of genetranscription (31). The second hypothesis is supported by studiesshowing that the activity of coactivators can be regulated byprotein kinases. Phosphorylation of residue Ser184 is essentialfor inducible activation by the coactivator Bob-1 (49). CBPcontains a signal-regulated domain required for stimulationof transcription; phosphorylation of this domain is controlledby nuclear calcium and calcium/calmodulin-dependent proteinkinase IV (10, 20). The histone acetyltransferase activity ofCBP is also regulated upon phosphorylation by p44 MAPK/ERK1(1). We have shown that differential phosphorylation of ${alpha}$ NACcontrols its half-life (36) and its nuclear entry (37). It ispossible that differential phosphorylation might regulate itsDNA-binding activity. Indeed, computer-based analysis identifiesresidue Ser70 within the DNA-binding domain as a potential phosphoacceptorsite (data not shown).

Alternatively, ${alpha}$ NAC might occupy its cognate binding site evenin nonexpressing cells. In this scenario, posttranslationalmodifications such as phosphorylation could function to allow ${alpha}$ NAC to recruit transcriptional repressors that would maintainosteocalcin gene expression as silent. The induction of osteocalcinexpression would require a different pattern of posttranslationalmodifications of the ${alpha}$ NAC protein that could then function asa coactivator of osteocalcin transcription. Additional studiesare required to distinguish between these various possibilities.

The osteocalcin gene promoter has been extensively studied.It is modularly organized and contains several positive andnegative regulatory elements (26). Our results have identifiedyet another regulatory sequence within the proximal osteocalcinpromoter region, the ${alpha}$ NAC binding site. The down-regulation ofosteocalcin promoter activity measured when endogenous ${alpha}$ NAC levelsare reduced in osteoblastic cells confirms the physiologicalrelevance of ${alpha}$ NAC in the control of osteocalcin gene transcription.It will prove interesting to characterize the functional interplaybetween the various transcriptional regulators that controlosteocalcin promoter activity in differentiating osteoblasts.

ACKNOWLEDGMENTS

We thank Gérard Karsenty (Baylor College of Medicine)and Shoukat Dedhar (University of British Columbia) for providingexpression vectors and reporter plasmids. We are indebted toRenny T. Franceschi (University of Michigan) for his generousgift of the MC3T3-E1 subclone stably transfected with the osteocalcin-luciferasereporter and for tips on the ChIP assay. We are grateful toMcGill University and the Genome Quebec Innovation Centre forallowing repeated usage of the real-time PCR instrument. MarkLepik and Guylaine Bédard prepared the figures.

This work was supported by the Shriners of North America.

FOOTNOTES

* Corresponding author. Mailing address: Genetics Unit, Shriners Hospital for Children, 1529 Cedar Avenue, Montréal, Québec, Canada H3G 1A6. Phone: (514) 282-7155. Fax: (514) 842-5581. E-mail: rst-arnaud{at}shriners.mcgill.ca.

Present address: Aventis Pharma Deutschland GmbH, IndustrieparkHoechst, D-65926 Frankfurt am Main, Germany.

REFERENCES

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