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Originally published In Press as doi:10.1074/jbc.M510514200 on April 4, 2006

J. Biol. Chem., Vol. 281, Issue 23, 16090-16098, June 9, 2006
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NF-Y and CCAAT/Enhancer-binding Protein {alpha} Synergistically Activate the Mouse Amelogenin Gene*Formula

Yucheng Xu{ddagger}, Yan Larry Zhou{ddagger}, Wen Luo{ddagger}, Qin-Shi Zhu§, Daniel Levy§, Ormond A. MacDougald, and Malcolm L. Snead{ddagger}1

From the {ddagger}Center for Craniofacial Molecular Biology and §Department of Biochemistry and Molecular Biology, University of Southern California, Los Angeles, California 90033 and Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0622

Received for publication, September 26, 2005 , and in revised form, February 10, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Amelogenin is the major protein component of the forming enamel matrix. In situ hybridization revealed a periodicity for amelogenin mRNA hybridization signals ranging from low to high transcript abundance on serial sections of developing mouse teeth. This in vivo observation led us to examine the amelogenin promoter for the activity of transcription factor(s) that account for this expression aspect of the regulation for the amelogenin gene. We have previously shown that CCAAT/enhancer-binding protein {alpha} (C/EBP{alpha}) is a potent transactivator of the mouse X-chromosomal amelogenin gene acting at the C/EBP{alpha} cis-element located in the –70/+52 minimal promoter. The minimal promoter contains a reversed CCAAT box (–58/–54) that is four base pairs downstream from the C/EBP{alpha} binding site. Similar to the C/EBP{alpha} binding site, the integrity of the reversed CCAAT box is also required for maintaining the activity of the basal promoter. We therefore focused on transcription factors that interact with the reversed CCAAT box. Using electrophoretic mobility shift assays we demonstrated that NF-Y was directly bound to this reversed CCAAT site. Co-transfection of C/EBP{alpha} and NF-Y synergistically increased the promoter activity. In contrast, increased expression of NF-Y alone had only marginal effects on the promoter. A dominant-negative DNA binding-deficient NF-Y mutant (NF-YAm29) dramatically decreased the promoter activity both in the absence or presence of exogenous expression of C/EBP{alpha}. We identified protein-protein interactions between C/EBP{alpha} and NF-Y by a co-immunoprecipitation analysis. These results suggest that C/EBP{alpha} and NF-Y synergistically activate the mouse amelogenin gene and can contribute to its physiological regulation during amelogenesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Tooth organogenesis is a complex developmental process that is dependent upon a series of reciprocal and instructive signals (1). These signals culminate to orchestrate expression of the amelogenin gene, the major organic component of the enamel matrix. Amelogenin plays a key role in regulating proper enamel mineralization and is believed to regulate its own replacement by the mineral phase to create a woven hierarchical architecture that accounts for the unique material properties of enamel (2–5). Mutations to the human amelogenin gene have been linked to patients with the inherited enamel defect X-linked amelogenesis imperfecta (6).

Amelogenin is specifically expressed and secreted by ameloblast cells, and this process is tightly controlled spatiotemporally (7). The murine amelogenin promoter has been isolated from the mouse X-chromosome. Based upon transgenic mouse studies, our laboratory has demonstrated that the 2,263 nucleotides upstream of amelogenin start codon fully recapitulate the endogenous amelogenin gene expression profile within time and space (8). Deletion analysis of this 2,263-nucleotide stretch demonstrates that the –70/+52-bp minimal promoter is indispensable for maintaining transcriptional activity. In addition, this minimal promoter contains a CCAAT/enhancer-binding site that is required for both basal promoter activity and C/EBP{alpha}-mediated transactivation (9). C/EBPs2 (CCAAT/enhancer-binding proteins) are a family of transcription factors that include a highly conserved, basic leucine zipper domain at the C terminus for dimerization and DNA binding, and they function in regulating cellular differentiation in multiple tissues (10–17). At least six members of the family have been isolated and characterized, and they have been named {alpha}, beta, {gamma}, {delta}, {epsilon}, and {zeta} (18–23).

Sequence analysis of the amelogenin minimal promoter (–70/+52) also revealed a reversed CCAAT box located in the –58/–54 region, four base pairs downstream to the C/EBP{alpha} binding site. Several transcription factors are able to recognize the CCAAT box, such as CCAAT transcription factor/nuclear factor 1 (24–26), CCAAT displacement protein (27–30), C/EBP (20, 31, 32), and NF-Y (nuclear factor-Y) (33). Among these transcription factors, NF-Y was regarded as a leading candidate based on the following. First, of all the potential CCAAT-binding proteins, only NF-Y has been shown to be absolutely required for all CCAAT pentanucleotide bona fide sequences (33–36). Second, several lines of evidence indicate that NF-Y cooperatively interacts with C/EBP{alpha} to function in transcriptional regulation on a variety of promoters (37–39). NF-Y is a heterotrimeric protein consisting of three subunits, NF-YA (also termed CCAAT binding factor B (CBF-B)), NF-YB (CBF-A), and NF-YC (CBF-C). Alignment of amino acid sequences of these three subunits reveals several highly conserved regions. NF-YA has a glutamine-rich region, a serine/threonine-rich region, a subunit interaction domain, and a DNA-binding domain. NF-YB contains a histone fold motif and a TATA-binding protein (TBP)-binding domain. Similar to NF-YB, NF-YC bears a histone fold motif, a TBP-binding domain, and an additional glutamine-rich region (40). All three subunits are necessary for DNA binding. The two subunits NF-YB and NF-YC first form a heterodimer via their histone fold motifs. The dimer then provides a suitable docking site for NF-YA to bind to form a functionally active NF-Y heterotrimeric protein (41).

In this study, we have demonstrated that NF-Y is directly bound to the –58/–54-bp CCAAT box within the amelogenin promoter. Moreover, NF-Y and C/EBP{alpha} synergistically increase the minimal amelogenin promoter activity, although NF-Y alone has only marginal effects on the promoter. We also identified protein-protein interactions between NF-Y and C/EBP{alpha}. Finally, ameloblasts from developing mouse teeth in vivo express both C/EBP{alpha} and NF-YA during development at a stage corresponding to maximum amelogenin expression. Taken together, these results suggest that NF-Y and C/EBP{alpha} synergistically activate the mouse amelogenin gene and could contribute to the physiologic regulation of amelogenin expression during enamel formation in vivo.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
In Situ Hybridization—In situ hybridization was used to determine the precise spatial distribution for amelogenin mRNA from lower incisors. An antisense amelogenin RNA probe was labeled by synthesis in the presence of [35S]thiophosphate-UTP (PerkinElmer Life Sciences) to specific activity of ~5 x 108 cpm/µg of RNA. The in situ hybridization method has been previously published and used without modification (7).

Preparation of Chicken Anti-C/EBP{alpha} Antibody—A C/EBP{alpha} internal epitope peptide (amino acids 249–263) was synthesized at the Microchemical Core Facility (University of Southern California Microchemical Core Facility) and used to generate chicken polyclonal antibodies that were purified from the yolk by affinity chromatography to the immobilized peptide.

Immunohistochemistry—Immunostaining was performed according to Couwenhoven et al. (42), using an anti-C/EBP{alpha} chicken polyclonal antibody (1:200; Invitrogen) or a commercially available polyclonal antibody to NF-YA (1:200, sc-10779; Santa Cruz Biotechnology, Santa Cruz, CA), followed by appropriate horseradish peroxidase-conjugated secondary antibodies (1:500; Zymed Laboratories Inc., San Francisco, CA). A substrate-chromogen mixture (Zymed Laboratories) was used for signal detection.

Plasmids and Cell Culture—The reporter constructs of p70-luc, mC/EBP{alpha}-p70-luc, and p51-luc were prepared as previously described (9). To generate mp70-luc and mC/EBP{alpha}-mp70-luc, the promoter regions were prepared by polymerase chain reactions with p70-luc as the template using a common 3'-primer (5'-TATTCTCGAGTGTATGCTCAGTGAG-3'; the XhoI site is underlined) and respective 5'-primers (5'-CGTGCTAGCTCAGAAACCTGATCAGCTGTTCAAA-3'; and 5'-CGTGCTAGCTTCAGTCTAGAGATCAGCTGTTCAAA-3'; the NheI sites are underlined and the mutated site is in boldface). The PCR products were digested with NheI and XhoI and inserted 5' to 3' into the NheI-XhoI site of pGL3-Basic (Promega, Madison, WI) and verified by nucleotide sequence analysis. The expression vector for C/EBP{alpha} was described previously (43). The expression vectors for three subunits of NF-Y (NF-YA, NF-YB, NF-YC) (44) were provided by Dr. Hiroyoshi Ariga (Hokkaido University, Hokkaido, Japan). The dominant-negative NF-YA expression vector (NF-YAm29) (45) was provided by Dr. Timothy Osborne (University of California at Irvine, Irvine, CA). A mouse ameloblast-like cell line LS8 was maintained as previously described (9).

Transient Transfection and Luciferase Assay—Variable amounts of plasmid DNA were used for transient transfection for each well of 12-well plates. The amounts of DNA varied based upon experimental conditions and are documented in the figure legends. To normalize transfection efficiency, 75 ng of pCMV-lacZ/well was co-transfected as an internal control. The day before transfection, LS8 cells were plated in 12-well plates so that they were 50–80% confluent at the time of transfection. At the time of plating and during transfection, antibiotics were avoided. Three hours before transfection, cells were washed twice with Dulbecco's modified Eagle's medium and subsequently cultured in serum-free Dulbecco's modified Eagle's medium. Plasmid DNA (0.75 µg) was diluted into 62 µl of medium in a 5-ml Falcon culture tube, and 5 µl of Plus reagent (Invitrogen) was added, mixed, and incubated for 15 min at room temperature. In a second tube, 2.5 µl of Lipofectamine reagent (Invitrogen) was diluted into 62 µl of medium and mixed. The contents of these two tubes were combined, mixed, and incubated for another 15 min at room temperature. While complexes were forming, the medium on the cells was replaced with 0.5 ml of fresh medium. The DNA-Plus-Lipofectamine complex was added to each well of cells and mixed gently. The cells were incubated for 3 h at 37 °C and 5% CO2. After removal of the medium containing DNA-Plus-Lipofectamine complex, cells were incubated in 1 ml of fresh complete medium for an additional 22 h and were subjected to luciferase assay with a Dual-Light kit (Applied Biosystems, Foster City, CA).

Cells were washed twice with phosphate-buffered saline, pH 7.4, and lysed in 100 µl of lysis buffer (100 mM potassium phosphate, pH 7.8, 0.2% Triton X-100, 0.5 mM dithiothreitol). Cell scrapers (Corning Inc., Acton, MA) were used to detach cells from the plate. Cell lysates were transferred to a microcentrifuge tube and centrifuged at maximum speed for 2 min. The extracts (supernatant) were transferred to a fresh tube and stored at –70 °C. At the time of chemiluminescent detection, buffers A and B (Applied Biosystems) were equilibrated to room temperature, and Galacton-Plus Substrate was added to buffer B at the ratio 1:100. A 10-µl aliquot of cell extracts was transferred to a luminometer tube in which 25 µl of buffer A was added, immediately followed by the addition of 100 µl of buffer B. After a 15-s delay, the luciferase signal was obtained for 5 s in a luminometer (Lumat, Berthold, ND). After 45 min of incubation at room temperature, 100 µl of Accelerator-II was added, and beta-galactosidase signal was measured for 5 s in the same luminometer.

Preparation of Nuclear Extracts—LS8 cells (100-mm plates) were transiently transfected with 10 µg of C/EBP{alpha} expression vector by the calcium phosphate co-precipitation method. Briefly, 10 µg of DNA was mixed with 62 µl of 2 M CaCl2, diluted to 500 µl with double distilled H2O, and added dropwise to the 37 °C prewarmed 2x HBS (HEPES-buffered saline) solution. After standing for 20 min at room temperature, the DNA-calcium phosphate co-precipitates were added dropwise to the surface of the medium covering the cells and incubated overnight. The next day, medium containing the calcium phosphate was removed and replaced with fresh complete medium for an additional 24 h. LS8 cells were washed twice with cold phosphate-buffered saline, pH 7.4, and scraped off in 1 ml of hypotonic buffer (20 mM Hepes, pH 7.6, 10 mM KCl, 1 mM MgCl2, 0.1% Triton X-100, 20% glycerol, 2 mM phenylmethylsulfonyl fluoride, 5 µl/ml of aprotinin (Sigma), 5 µg/ml of leupeptin, 0.5 mM dithiothreitol). Cell lysates were Dounce homogenized for 15 strokes with a type A pestle on ice, transferred to a 1.5-ml tube, and centrifuged at 3,000 rpm for 5 min at 4 °C. The pellet was resuspended in 100 µlof cold extraction buffer (20 mM Hepes, pH 7.6, 10 mM KCl, 1 mM MgCl2, 0.1% Triton X-100, 20% glycerol, 2 mM phenylmethylsulfonyl, 5 µl/ml of aprotinin (Sigma), 5 µg/ml of leupeptin, 0.5 mM dithiothreitol, 420 mM NaCl) and mixed on a rotator for 1 h at 4 °C. Nuclear debris was pelleted by centrifugation at 15,000 rpm for 10 min at 4 °C. Supernatants containing the nuclear extracts were frozen in liquid N2 and stored at –80 °C. The protein concentration was determined using a Bio-Rad protein assay kit with bovine serum albumin as the standard.


Figure 1
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  		FIGURE 1.
In situ hybridization analysis of amelogenin mRNA abundance in a 1-day postnatal mouse mandibular incisor. A radioactive antisense amelogenin RNA probe was hybridized to serial tissue sections to detect sense amelogenin transcripts (mRNA). The images show variation in grain density following radioautography. The variation (panels A–D) in grain density corresponding to mRNA abundance in serial sections is shown in the lower chart and reveals a periodicity among adjacent ameloblasts for the minima (A) and maxima (D) of transcript abundance.

 
Electrophoresis Mobility Shift Assay (EMSA)—The oligodeoxynucleotide probes were synthesized, annealed, and radiolabeled with {alpha}-32P (PerkinElmer) using the fill-in reaction and Klenow enzyme (Roche Applied Science). A mass of 8–15 µg of nuclear extract was added from selected samples in the EMSA buffer at the final concentration of 40 mM Tris-Cl, pH 7.9, 12 mM MgCl2, 60 mM KCl, 2 mM EGTA, 2 mM EDTA, 25% glycerol, 0.2 µg/µl of poly(dI-dC)·poly(dI-dC) and incubated for 2 h at 4 °C. Radiolabeled oligonucleotides (~3,000–6,000 cpm/fmol) in 1.5-µl volume were added and incubated for an additional 1 h at room temperature. Where indicated, 50-fold molar excess of unlabeled competitor probes, antibodies (1 µg for anti-NF-Y, and 2 µg for anti-C/EBP{alpha}, Santa Cruz Biotechnology), or normal serum was included in the binding reaction prior to the addition of radiolabeled probes. After mixing with 1 µl of loading buffer containing 250 mM Tris-Cl, pH 7.5, 0.2% bromphenol blue, 0.2% xylene cyanol, and 40% glycerol, the mixture was resolved on a 5% nondenaturing polyacrylamide gel in 0.5x TBE buffer prerun at 20 mA for at least 30 min. Gel electrophoresis was carried out at 30 mA for 2.5 h. The gel was dried, and bands were visualized by autoradiography. The sequences of the oligonucleotides were as follows: wild-type (WT) sense strand, 5'-TTTTTCATTCAGAAACCTGATTGGCTGTTC-3'; wild-type (WT) antisense strand, 5'-GAACAGCCAATCAGGTTTCTGAATG-3'; mutant C/EBP{alpha} (mC/EBP{alpha}) sense strand, 5'-TTTTTCATTCAGTCTAGAGATTGGCTGTTC-3'; mutant C/EBP{alpha} antisense strand, 5'-GAACAGCCAA TCTCTAGACTGAATG-3'; mutant NF-Y (mNF-Y) sense strand, 5'-TTTTTCATTCAGAAACCTGATCAGCTGTTC-3'; mutant NF-Y antisense strand, 5'-GAACAGCTGATCAGGTTTCTGAATG-3'; mutant C/EBP{alpha}-mutant NF-Y (mC/EBP{alpha}-mNF-Y) sense strand, 5'-TTTTTCATTCAGTCTAGAGATCAGCTGTTC-3'; mutant C/EBP{alpha}-mutant NF-Y antisense strand, 5'-GAACAGCTGATCTCTAGACTGAATG-3' (mutated nucleotides are in boldface).


Figure 2
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  		FIGURE 2.
Antibody detection of C/EBP{alpha} (A, B) and NF-YA (C, D) expression pattern in serial sections from mouse mandibular incisors engaged in amelogenin expression. Immunohistochemical signals are shown by the precipitated chromogen.

 
Co-immunoprecipitation Assay and Western Blotting—LS8 cells, ~90% confluent in a 100-mm cell culture plate, were washed twice in ice-cold phosphate-buffered saline. Ice-cold radioimmune precipitation buffer (1 ml of 1x phosphate-buffered saline, 1% Nonidet P-40, 0.1 mg/ml of phenylmethylsulfonyl fluoride, 30 µl/ml of aprotinin (Sigma), and 1 mM sodium orthovanadate) was added, and the cells were collected using a cell scraper (Corning Inc.) and lysed by passing six times through a 22-gauge needle at 4 °C. After centrifugation at 3,000 rpm for 15 min at 4 °C, the protein concentration of the supernatant was measured using a Bio-Rad protein assay kit with bovine serum albumin as standards. For immunoprecipitation, 500 µg of total protein was precleaned with 50 µl of protein G-agarose beads (Sigma) prior to the addition of 2 µg of primary antibody (Santa Cruz Biotechnology) for overnight incubation at 4 °C with rotation. Protein G-agarose beads (20 µl; IgG binding capacity at 10–20 µg/µl) were added and incubated for 2 h at 4 °C with rotation. Immunoprecipitates were collected by centrifugation at 2,500 rpm for 5 min at 4 °C. The pellets were washed three times with 1 ml of phosphate-buffered saline. After the final wash, the pellets were resuspended in an equal volume of 2x SDS loading buffer, boiled for 5 min, and stored at –70 °C. Samples were resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to Immobilon-P membrane (Millipore Corp., Billerica, MA). The membranes were incubated in blocking buffer overnight at 4 °C, followed by incubation with the primary antibody for 1 h and the appropriate horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences) for 1 h at room temperature. Protein-antibody complexes were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences).


Figure 3
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  		FIGURE 3.
The integrity of the C/EBP{alpha} binding site and reversed CCAAT box is required for maintaining the activity of the basal amelogenin promoter and for C/EBP{alpha}-mediated transactivation. A, nucleotide sequence alignment of the amelogenin promoter. The –80/–40 region of the murine X-chromosomal amelogenin promoter is aligned among corresponding regions of the pig, bovine, horse, monkey, and human amelogenin promoters. The C/EBP{alpha} consensus sequence is shown as TNNNGNAA in boldface. A consensus pentanucleotide sequence ATTGG is also shown in boldface. B, schematic representation of the 5'-proximal region of the mouse amelogenin promoter in the luciferase reporter construct. The nucleotide sequence of –70/–51 is shown in which the C/EBP{alpha} binding site and the CCAAT box are included. The CCAAT box (–58 to –54) is represented by a "C" in the diagram. The p70-luc represents a wild-type minimal amelogenin promoter reporter construct; mC/EBP{alpha}-p70-luc represents a mutated C/EBP{alpha} binding site with a wild-type CCAAT box; mp70-luc represents a wild-type C/EBP{alpha} binding site with a mutated CCAAT box; mC/EBP{alpha}-mp70-luc represents mutations to both the C/EBP{alpha} binding site and the CCAAT box; p51-luc represents deletion of the nucleotide stretch containing both the C/EBP{alpha} site and the CCAAT box up to the –51-bp position of the promoter. The mutated nucleotides are shown in lowercase with boldface. C, results of transient transfection experiments. 250 ng of various reporter constructs (p70-luc, mp70-luc, mC/EBP{alpha}-p70-luc, mC/EBP{alpha}-mp70-luc, and p51-luc) were transiently transfected into LS8 cells with 200 ng of C/EBP{alpha} expression plasmid or empty vector pcDNA3. In all cases, pCMV-lacZ (75 ng) was included as an internal control for transfection efficiency. The relative luciferase activity was calculated by normalizing luciferase activity with beta-galactosidase activity. The mean ± S.D. from at least three independent experiments is shown, and the level of p70-luc in the absence of exogenous C/EBP{alpha} is set arbitrarily as 1.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Amelogenin mRNA Abundance in Mouse Mandibular Incisors—In situ hybridization studies have previously demonstrated that amelogenin mRNA level is regulated in a spatial- and temporal-restricted pattern during mouse molar tooth organogenesis (7). In this report, we have investigated the expression pattern of mouse amelogenin mRNA in lower incisors. In situ hybridization data revealed a periodicity for amelogenin mRNA by ameloblasts, ranging from low (Fig. 1A) to high (Fig. 1D) transcript abundance on serial sagittal sections of developing mouse incisors. This finding from mouse incisors, combined with our previous observation from mouse molars (7), suggests that amelogenin expression levels are tightly regulated at the temporal and spatial level in vivo to accommodate the process of enamel biomineralization (5).

Immunohistochemical Localization of C/EBP{alpha} and NF-YA in Mouse Mandibular Incisors—We have previously identified C/EBP{alpha} as a transactivator of amelogenin gene in vitro (9). To investigate the relationship of C/EBP{alpha} to amelogenin expression in vivo, we performed immunohistochemistry to detect C/EBP{alpha}. Consistent with the amelogenin mRNA hybridization signal pattern, immunohistochemistry for C/EBP{alpha} protein revealed a repeating periodicity for C/EBP{alpha} with subcellular localization to either the cytoplasm or the nucleus of ameloblast cells in the mandibular incisors (Fig. 2, A and B). Moreover, the anti-NF-YA antibody localized to the nucleus of ameloblasts from mandibular incisors (Fig. 2, C and D). The pattern for amelogenin mRNA abundance and C/EBP{alpha}/NF-YA localization within ameloblasts suggested to us a potential role for C/EBP{alpha}/NF-Y in the regulation of amelogenin gene expression in vivo. We pursued the role for these transcription factors to regulate the amelogenin promoter in vitro using an ameloblast-like cell line, LS8.

The Minimal Amelogenin Promoter Activity Is Dependent on Both the –70/–63-bp C/EBP{alpha} Cis-element and the –58/–54-bp CCAAT Box—Deletion analysis of the mouse amelogenin promoter has shown that the –70/+51-bp region functions as a minimal promoter in LS8 cells (9). A C/EBP{alpha} cis-element is identified in the –70/–63 bp, and it is through this region that C/EBP{alpha} up-regulates the promoter activity in a dose-dependent manner (9). Sequence alignment of the –80/–40 bp of X-chromosomal amelogenin gene from mouse, pig, bovine, horse, monkey, and human revealed a highly conserved reversed CCAAT box located in the –58/–54-bp region (Fig. 3A). All sequences showed a match for the pentanucleotides of ATTGG, a reversed CCAAT box. To investigate the potential role of this cis-element in the regulation of amelogenin promoter, the ATTGG sequence in the p70-luc reporter construct was mutated to ATCAG to generate the "mp70-luc" construct (Fig. 3B). Transient transfection of mp70-luc into LS8 cells, compared with that of p70-luc, showed a complete loss of basal promoter activity (Fig. 3C, lane 2). This suggested an important role for the CCAAT box in maintaining the activity of the basal amelogenin promoter. Sequence alignment also revealed that the C/EBP{alpha} binding site was close to the CCAAT box, just four base pairs upstream to the CCAAT box (Fig. 3A). To further understand the role of the C/EBP{alpha} binding site and the CCAAT box in regulating the amelogenin promoter and their potential relationship with each other, a series of mutation constructs were generated as shown in Fig. 3B. Each of these mutation constructs, mC/EBP{alpha}-p70-luc (mutated C/EBP{alpha} site), mp70-luc (mutated CCAAT site), mC/EBP{alpha}-mp70-luc (mutated C/EBP{alpha} site and mutated CCAAT site), and p51-luc (deleted C/EBP{alpha} and deleted CCAAT sites) or their wild-type counterpart, was co-transfected into LS8 cells with or without the C/EBP{alpha} expression vector and analyzed in transient transfection assays. The mutation of the C/EBP{alpha} cis-element (mC/EBP{alpha}-p70-luc), CCAAT box (mp70-luc), dual mutations (mC/EBP{alpha}-mp70-luc), as well as the dual deleted C/EBP{alpha} and CCAAT box construct (p51-luc), all showed a complete loss of the basal promoter activity (Fig. 3C, lanes 2–5). Furthermore, in response to the exogenous expression of C/EBP{alpha}, the reporter activity of wild-type construct (p70-luc) was increased dramatically (Fig. 3C, lane 6), whereas the reporter activity for the remaining four altered C/EBP{alpha} and/or CCAAT box constructs was greatly reduced (lanes 7–10). The observed residual activity (lanes 7–10) may be due to other factors, induced by overexpression of C/EBP{alpha}, nonspecifically affecting the amelogenin promoter. Taken together, these data indicated that the integrity of the C/EBP{alpha} cis-element and the reversed CCAAT box was required to maintain the basal promoter activity and C/EBP{alpha}-mediated transactivation.


Figure 4
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  		FIGURE 4.
EMSA of the C/EBP{alpha} and the CCAAT box cis-elements. A, the sequence is shown of the –77/–48 region of the amelogenin promoter containing both C/EBP{alpha} and CCAAT box sites used as the probe in EMSA. The C/EBP{alpha} binding site is in boldface. The CCAAT box is underlined and in boldface. B, nuclear extracts were prepared from LS8 cells transfected with the C/EBP{alpha} expression plasmid (NE/{alpha}). EMSA was performed by adding the 32P-labeled probe with or without nuclear extracts incubated for 1 h at room temperature, analyzed on a 5% nondenaturing polyacrylamide gel, and visualized by autoradiography. Prior to addition of the probe, the reaction mixture was incubated where indicated with unlabeled competitor, or antibodies, or normal goat or rabbit serum for 2 h at 4 °C. Shown in the lane indicated is the binding of the probe in the absence of nuclear extract (–)(lane 1), with nuclear extract (+)(lanes 2–7), and additional 50-fold molar excess of unlabeled probe (lane 3), NF-Y antibody (lane 4; sc-7712x; Santa Cruz Biotechnology), normal goat serum (lane 5), C/EBP{alpha} antibody (lane 6; sc-61; Santa Cruz Biotechnology), and normal rabbit serum (lane 7).

 
NF-Y Is Directly Bound to the CCAAT Box in the Minimal Amelogenin Promoter—Although a number of transcription factors are reported to recognize the CCAAT motif (20, 24, 27, 33), subsequent analyses reveal that there is divergence in the recognition sequence of CCAAT and that only NF-Y exclusively requires the bona fide CCAAT pentanucleotide (33–36). To test whether NF-Y was able to bind to this reversed CCAAT box in the minimal amelogenin promoter, we performed EMSA using a probe bearing the C/EBP{alpha} cis-element and the CCAAT motif shown in Fig. 4A. As shown in Fig. 4B, DNA-protein complexes formed (lane 2), and the complex could be inhibited by the addition of a 50-fold molar excess of unlabeled probe (lane 3). A rabbit antibody specific to C/EBP{alpha} was able to supershift the DNA-protein complex (lane 6), a finding consistent with our previous study (9), whereas normal rabbit serum failed to supershift the complex (lane 7). An anti-NF-Y goat antibody supershifted a substantial amount of the formed EMSA complex (lane 4), whereas no supershift was observed with the addition of normal goat serum (lane 5). These data demonstrated that NF-Y was a major component of the DNA-protein complex.


Figure 5
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  		FIGURE 5.
Mutational analysis of C/EBP{alpha} and NF-Y proteins binding to the –77/–48 probe. A, the sequences are shown of the wild-type (Wt) and mutated (mC/EBP{alpha}, mNF-Y, and mC/EBP{alpha}-mNF-Y) probes. The C/EBP{alpha} binding site is in bold face, and the CCAAT box is underlined and in bold face. The mutated nucleotides are shown in lower-case. B, EMSA analysis was performed as in Fig. 4B. Autoradiogram from EMSA with the binding of WT probe (lanes 1–5), binding of mC/EBP{alpha} probe (lanes 6–10), binding of mNF-Y probe (lanes 11–15), and binding mC/EBP{alpha}-mNF-Y probe (lanes 16–20).

 
Because there are only four nucleotides between these two binding sites, it is possible that the binding of one protein may interfere with the binding of the other. To investigate this possibility, four different probes were designed, each with a different combination of wild-type or mutated binding sites for C/EBP{alpha} or NF-Y (Fig. 5A). The effects of these different probes on the C/EBP{alpha} and NF-Y binding were assessed by EMSA as shown in Fig. 5B. As expected, a shifted band formed with wild-type probe (lane 2), and both anti-NF-Y and anti-C/EBP{alpha} antibodies were able to recognize their respective transcription factor to form supershift bands (lanes 4 and 5). In contrast, the mC/EBP{alpha} probe, in which the C/EBP{alpha} site was mutated and NF-Y site was wild-type, resulted in the loss of supershifted band in the presence of anti-C/EBP{alpha} antibody (lane 10). However, the addition of anti-NF-Y antibody was still able to supershift the NF-Y-DNA complex (lane 9). When utilizing the mNF-Y probe bearing a mutated NF-Y site and a wild-type C/EBP{alpha} binding site, the intensity of shifted band was diminished (lane 12), and as expected, an anti-NF-Y antibody failed to supershift the complex (lane 14). Interestingly, an anti-C/EBP{alpha} antibody was not only able to supershift the DNA-protein complex but increased the intensity of the supershifted band as well (lane 15). This suggested that binding of NF-Y proteins impaired the binding of C/EBP{alpha} to the probe. Once NF-Y no longer recognized its cognate site in the mNF-Y probe, this absence allowed C/EBP{alpha} to form a more stable complex. When a probe with both the C/EBP{alpha} and NF-Y sites was mutated, no shifted band formed (lane 17) nor was a supershifted band observed with the addition of either an anti-NF-Y or an anti-C/EBP{alpha} antibody (lanes 19 and 20). The shifted bands of lower molecular weight lying below the primary shifted products are considered as noise and could result from other unknown factor(s) interacting with the probe.


Figure 6
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  		FIGURE 6.
C/EBP{alpha} and NF-Y synergism on the minimal amelogenin promoter. LS8 cells were transiently transfected with 250 ng of p70-luc reporter construct in the presence of 200 ng of empty vector (lane 1), expression vector for C/EBP{alpha} (lane 2), NF-Y (lane 3), dominant-negative form of NF-Y, NF-YAm 29 (lane 4), C/EBP{alpha} and NF-Y (lane 5), and C/EBP{alpha} and NF-YAm 29 (lane 6). pCMV-lacZ plasmid (75 ng) was included in all experiment groups as an internal control for transfection efficiency. Data reflect the mean ± S.D. of three independent experiments, with the response level of p70-luc in the absence of exogenous C/EBP{alpha} set arbitrarily as 1.

 


Figure 7
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  		FIGURE 7.
Co-immunoprecipitation of NF-YA and C/EBP{alpha} in LS8 cells. 2µg of C/EBP{alpha} expression vector was co-transfected into LS8 cells with 2µg each of NF-YA, NF-YB, and NF-YC expression vectors in a 100-mm cell culture plate. Whole cell lysates were prepared and immunoprecipitated (IP) with either an anti-NF-YA (sc-7712x; Santa Cruz Biotechnology) or an anti-C/EBP{alpha} antibody (sc-61x; Santa Cruz Biotechnology) and protein G-agarose beads (P-7700; Sigma). The immunoprecipitates or the total cell lysates were analyzed by SDS-PAGE, followed by immunoblot (IB) with antibodies against NF-YA (sc-10779; Santa Cruz Biotechnology) or C/EBP{alpha} (sc-61; Santa Cruz Biotechnology). Total cell lysates of 20 µg were immunoblotted with an antibody against NF-YA (lane 1) or C/EBP{alpha} (lane 4). Cell lysates were immunoprecipitated with an anti-NF-YA antibody, followed by immunoblotting with an anti-NF-YA (lane 2) or an anti-C/EBP{alpha} (lane 3) antibody. Cell lysates were immunoprecipitated with an anti-C/EBP{alpha} antibody, followed by immunoblotting with an anti-C/EBP{alpha} (lane 5) or an anti-NF-YA (lane 6) antibody.

 
NF-Y and C/EBP{alpha} Synergistically Activate the Mouse Amelogenin Promoter—To investigate the functional relationship between NF-Y and C/EBP{alpha} in regulating the minimal amelogenin promoter activity, NF-Y and C/EBP{alpha} expression plasmids were co-transfected with the p70-luc reporter construct into LS8 cells. Alternatively, a mutant form of NF-YA, termed NF-YAm 29, was also tested in this assay. NF-YAm 29, which has three amino acid substitutions (R311 -> A, G312 -> A, and E313 -> A) at its C terminus, is devoid of DNA binding capacity and acts as a dominant-negative mutant by sequestering the NF-YB/YC heterodimer into a defective NF-Y complex (45). As shown in Fig. 6, C/EBP{alpha} in isolation increased the promoter activity 7-fold (lane 2), whereas exogenous expression of wild-type NF-Y in isolation had only marginal effects on the promoter (lane 3). Co-transfection of C/EBP{alpha} with NF-Y served to synergistically increase the promoter activity to 16-fold (lane 5), a level that was two times greater than that of C/EBP{alpha} only. Furthermore, the presence of the dominant-negative mutant NF-YAm 29 dramatically decreased the promoter activity, either in the absence or the presence of exogenous C/EBP{alpha} expression (lanes 4 and 6). Theses observations demonstrated that NF-Y and C/EBP{alpha} synergistically activated the minimal amelogenin promoter activity, although by itself NF-Y exhibited only a marginal effect on the minimal amelogenin promoter activity.

Protein to Protein Interaction between NF-Y and C/EBP{alpha} in LS8 Cells—To examine whether NF-Y was able to interact with C/EBP{alpha} at a protein-protein level, we performed co-immunoprecipitation analysis in LS8 cells. A C/EBP{alpha} expression construct was co-transfected with NF-YA/YB/YC expression constructs into LS8 cells. NF-YA and C/EBP{alpha} were both detected in the LS8 cell lysates by Western blot with their respective antibodies (Fig. 7, lane 1 and lane 4). When immunoprecipitated with an anti-NF-YA antibody, NF-YA proteins were readily detected (lane 2). The precipitated complex also contained C/EBP{alpha}, which was co-immunoprecipitated efficiently with NF-YA (lane 3). The ability of the anti-NF-YA antibody to pull down C/EBP{alpha} demonstrated a protein-protein interaction between NF-YA and C/EBP{alpha}. The reciprocal experimental strategy was also performed and confirmed that a protein complex "pulled down" by an anti-C/EBP{alpha} antibody contained both C/EBP{alpha} and NF-YA proteins (lanes 5 and 6).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, in situ hybridization revealed a distinctive periodicity for amelogenin mRNA abundance among ameloblasts from mouse incisors at a developmental stage corresponding to maximum expression of amelogenin. Amelogenin is the most abundant protein in developing mammalian enamel and is believed to control the habit of the hydroxy-apatite mineral phase (2–6). This in vivo observation prompted us to examine the amelogenin promoter for transcription factors that could significantly alter amelogenin transcript abundance. Immunohistochemistry also revealed a repeating periodicity for C/EBP{alpha} and NF-YA localization within ameloblasts, suggesting a tight relationship for these transcription factors to participate in regulating amelogenin expression in vivo. These observations were the impetus for investigating the role of C/EBP{alpha} and NF-Y in regulating the amelogenin promoter in vitro.

We identified highly conserved C/EBP{alpha} and NF-Y binding motifs in the X-chromosomal amelogenin promoter among several mammalian species (Fig. 3A). The integrity of both transcription factor binding motifs was critical for maintaining the activity of the basal promoter and C/EBP{alpha}-mediated transactivation (Fig. 3C). Both C/EBP{alpha} and NF-Y were able to recognize their cis-elements (Fig. 4B). In addition, C/EBP{alpha} and NF-Y formed a protein-protein interaction complex (Fig. 7). As a consequence, C/EBP{alpha} and NF-Y synergistically activated the amelogenin promoter (Fig. 6). In contrast, NF-Y alone had only marginal effects on the promoter.

C/EBP{alpha} serves as a critical transcription factor during adipocyte differentiation, inducing gene expression required for fat metabolism as well as leading to the arrest of cell division (14, 46–49). Although C/EBP{alpha} is a transcription factor, intriguingly its ability to arrest growth does not require its DNA binding activity but rather is mediated via protein-protein interactions with the cell cycle inhibitor p21 (50), the cyclin-dependent kinases cdk2 and cdk4 (51), or E2F (52). In addition, C/EBP{alpha} has been reported to stimulate promoter activity, not through its DNA binding capacity but through its interaction with NF-Y bound to the EPHX1 CCAAT box (39).

NF-Y is a major CCAAT binding transcription factor that has been found to function mostly through interaction with other transcription factors (53–59). One explanation for transcription factor pairing is that NF-Y requires its associate protein, and vice versa, to form a more stable complex that binds to the DNA. This may explain the mechanism of synergism observed between C/EBP{alpha} and NF-Y that serves to cooperatively activate the amelogenin promoter observed in this study. However, it is also reasonable to argue that the binding of C/EBP{alpha} and NF-Y to the amelogenin promoter is mutually exclusive because of the proximity of these two binding sites (four nucleotides apart). Here, EMSA data showed that both NF-Y and C/EBP{alpha} were able to bind to their cognate sites (Fig. 4B, lanes 4 and 6; Fig. 5B, lanes 4 and 5). Interestingly, the mNF-Y probe, in which the NF-Y site was mutated and C/EBP{alpha} site was wild type, resulted in increased intensity of the supershifted band by an anti-C/EBP{alpha} antibody (Fig. 5B, lane 15), suggesting that binding of NF-Y did affect the binding of C/EBP{alpha}. In contrast, the binding of C/EBP{alpha} did not impair the binding of NF-Y (Fig. 5B, lane 4). A mutant amelogenin reporter construct, mp70-luc, bearing a mutant NF-Y site and a wild-type C/EBP{alpha} site showed complete loss of the basal promoter activity (Fig. 3C, lane 2) and was also incapable of responding to the exogenous C/EBP{alpha} even with its intact C/EBP{alpha} cis-element (Fig. 3C, lane 7). This suggested that NF-Y is indispensable for C/EBP{alpha}-mediated transactivation of the mouse amelogenin gene.


Figure 8
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  		FIGURE 8.
A proposed model for the mechanism underlying the synergism between C/EBP{alpha} and NF-Y in activating the amelogenin promoter. NF-Y facilitates the function of C/EBP{alpha} to synergistically activate the mouse amelogenin promoter. The postulated activation domain of C/EBP{alpha} is represented by a domain marked with an "A"inthe diagram. See "Discussion" for details.

 
Based on our observations, we propose a model explaining the mechanism underlying the synergism between C/EBP{alpha} and NF-Y in regulating the amelogenin promoter that is shown in Fig. 8. In the presence of NF-Y bound to its cognate site, the space for C/EBP{alpha} to bind to its cognate site is limited due to proximity of these two binding motifs. The C/EBP{alpha} dimer adjusts its conformation to adopt a shape to fit the site, which may be favored by associating with NF-Y through protein to protein interactions (Fig. 7). Under this circumstance, the binding of C/EBP{alpha} to its cognate site is perturbed (Fig. 5B, lane 5) with a conformational change that allows C/EBP{alpha} domains to be exposed and hence to recruit additional transcriptional factors to ensure transcriptional synergism (Fig. 6, lane 5). In the absence of NF-Y, C/EBP{alpha} binds tightly to its cognate site (Fig. 5B, lane 15) but with a different conformation that results in diminished transcription activation (Fig. 3C, lane 7). In support of this model is the mouse serum albumin promoter that contains a C/EBP{alpha} site tightly juxtaposed to a binding site for NF-Y, and this arrangement leads to strong synergistic activation of the serum albumin promoter (37).

In conclusion, we have demonstrated that there is a functional synergism between C/EBP{alpha} and NF-Y in regulating the amelogenin gene in vitro. Both factors are required for transcriptional activation, and NF-Y is indispensable for the robust expression. Furthermore, a model is proposed to explain the mechanism underlying the synergism between C/EBP{alpha} and NF-Y. These data, together with our previous identification of Msx2 as a transcriptional repressor in regulating the mouse amelogenin gene expression (60), suggest that these factors may cooperatively contribute to proper amelogenin expression in a temporally and spatially regulated fashion during tooth formation.


   FOOTNOTES
 
* This work was supported by NIDCR, National Institutes of Health Grant DE-06988. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. Back

1 To whom correspondence should be addressed: CSA 142, CCMB, University of Southern California, 2250 Alcazar St., Los Angeles, CA 90033. Tel.: 323-442-3178; Fax: 323-442-2981; E-mail: mlsnead{at}usc.edu.

2 The abbreviations used are: C/EBP{alpha}, CCAAT/enhancer-binding protein {alpha}; NF-Y, nuclear factor-Y; EMSA, electrophoretic mobility shift assay. Back


   ACKNOWLEDGMENTS
 
We thank our colleagues at the University of Southern California Center for Craniofacial Molecular Biology and the Institute for Genetic Medicine for their support. We are grateful for the constructive comments from the anonymous reviewers who have improved the readability of this manuscript. We thank Dr. H. Ariga (Hokkaido University) and Dr. T. Osborne (University of California at Irvine) for the generous gift of reagents critical to this investigation.



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