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Originally published In Press as doi:10.1074/jbc.M702097200 on August 17, 2007

J. Biol. Chem., Vol. 282, Issue 41, 29882-29889, October 12, 2007
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CCAAT/Enhancer-binding Protein {delta} (C/EBP{delta}) Maintains Amelogenin Expression in the Absence of C/EBP{alpha} in Vivo*Formula

Yucheng Xu{ddagger}, Yan Larry Zhou{ddagger}, Frank J. Gonzalez§, and Malcolm L. Snead{ddagger}1

From the {ddagger}The Center for Craniofacial Molecular Biology, University of Southern California, Los Angeles, California 90033 and §Laboratory of Metabolism, NCI, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, March 12, 2007 , and in revised form, August 16, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
C/EBP{alpha} is implicated to regulate mouse amelogenin gene expression during tooth enamel formation in vitro. Because enamel formation occurs during postnatal development and C/EBP{alpha}-deficient mice die at birth, we used the Cre/loxP recombination system to characterize amelogenin expression in C/EBP{alpha} conditional knock-out mice. Mice carrying the Cre transgene under the control of the human keratin-14 promoter show robust Cre expression in the ameloblast cell lineage. Mating between mice bearing the floxed C/EBP{alpha} allele with keratin-14-Cre mice generate C/EBP{alpha} conditional knock-out mice. Real-time PCR analysis shows that removal of one C/EBP{alpha} allele from the molar enamel epithelial organ of 3-day postnatal mice results in dramatic decrease in endogenous C/EBP{alpha} mRNA levels and coordinately altered amelogenin mRNA abundance. Conditional deletion of both C/EBP{alpha} alleles further diminishes C/EBP{alpha} mRNA levels; however, rather than ablating amelogenin expression, we observe wild-type amelogenin mRNA abundance levels. We examined C/EBPbeta and nuclear factor YA expression, two transcription factors that had previously been shown to modestly participate in amelogenin expression, in vitro but found no significant changes in either of their mRNA abundance levels comparing conditional knock-out mice with wild-type counterparts. Although the abundance of C/EBP{delta} is also unchanged in C/EBP{alpha} conditional knock-out mice, in vitro we find that C/EBP{delta} activates the mouse amelogenin promoter and synergistically cooperates with nuclear factor Y, suggesting that C/EBP{delta} can functionally substitute for C/EBP{alpha} to produce an enamel matrix competent to direct biomineralization.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In vitro C/EBP{alpha}2 has been demonstrated to act as a strong transactivator for amelogenin and to synergize with NF-Y to further increase expression, whereas activation is opposed by Msx2 in a pathway working through protein to protein interactions (1–5). Mice homozygous for the C/EBP{alpha} gene deletion die with in 8 h of birth from either hypoglycemia (6) or impaired function of type II pneumocytes (7). This perinatal lethal phenotype has prevented our observation of the effects from the loss of C/EBP{alpha} on postnatal tooth formation as this is the precise developmental period when enamel formation occurs and amelogenin mRNA expression is robust (8, 9).

The Cre/LoxP recombination system offers a method to circumvent this problem and has been successfully applied to investigating the role of C/EBP{alpha} in energy metabolism in the liver and adipose tissues at later stages of postnatal development (10, 11). The homozygous C/EBP{alpha}-loxP (C/EBP{alpha}fl/fl) mice are indistinguishable from their wild-type counterparts (10).

The search for a promoter useful for driving Cre recombinase in ectoderm-derived ameloblasts identified the keratin 14 (K14) promoter (12–15). Therefore, we used the K14 promoter to drive Cre-mediated recombination of floxed C/EBP{alpha} loci predicted to result in the loss of amelogenin expression.

Here we report on the generation of C/EBP{alpha} conditional knock-out mice created by crossing mice bearing the C/EBP{alpha}fl/fl with K14-Cre mice. This conditional ablation allowed us to investigate the relationship between C/EBP{alpha} and amelogenin expression in vivo utilizing real-time PCR technique to measure mRNA levels. We find that in the absence of mRNA for C/EBP{alpha} that C/EBP{delta} is redundant, serving to maintain amelogenin expression at wild-type levels in C/EBP{alpha} conditional knock-out mice.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Transient Transfection and Luciferase Assay—Transient transfection and luciferase assays were performed as described previously (3).

Animal Preparation—The K14-Cre transgenic line, the R26R reporter line, and the C/EBP{alpha}fl/fl (fl, flanked by loxP sites) mouse stain have been described previously (10, 16, 17). Mating K14-Cre+/– with R26R+/– mice generated R26R;K14-Cre mice (double transgenic). Mating K14-Cre+/– with fl/fl (C/EBP{alpha}fl/fl) mice generated wt/fl;K14-Cre mice; subsequent mating between wt/fl;K14-Cre mice generated fl/fl;K14-Cre homozygous conditional knock-out mice for C/EBP{alpha}. For ease of identification, we refer to the wild-type animal as C/EBP{alpha}+/+, the wt/fl;K14-Cre animal as C/EBP{alpha}+/–, and the fl/fl;K14-Cre animal as C/EBP{alpha}–/– to allow the C/EBP{alpha} allele status to be easily tracked.

Genotyping of Wild-type (+), loxP-targeted (fl), and Cre-mediated Recombination (–) for C/EBP{alpha} Alleles—Genomic DNA from mouse tails was isolated by digestion in a buffer containing 0.6 mg/ml proteinase K, 50 mM Tris-Cl, pH 8.0, 100 mM EDTA, and 0.5% SDS at 55 °C overnight. The solution was subjected to extraction with phenol, phenol/chloroform, and chloroform. DNA in the aqueous phase was precipitated by the addition of 2 volumes of ethanol. An additional wash step in 70% ethanol was essential to remove traces of SDS and phenol before biochemical manipulation.

As shown in Fig. 1A, PCR primers F4, (5'-AACCTCCACCTCCCCTCG-3'), F6 (5'-TCTGATGCCGCCGTGTTC-3'), F7 (5'-CTCCAGTGTGGTCTGTGTTGG-3'), B4 (5'-GCCAAACCCCGTGTTCAC-3'), and B6 (5'-CCCCTGATGCTCTTCGTCCAG-3') were used to differentiate the wild-type, floxed C/EBP{alpha}, and knock-out C/EBP{alpha} allele. The F7/B4 primer pairs were used to detect the 600-bp wild-type allele, the F6/B6 primer pairs were used to detect the 400-bp floxed C/EBP{alpha} allele, and the F4/B4 primer pairs were used to detect the 900-bp knock-out allele (Fig. 1A). The primer pair used to identify the 700-bp Cre allele was: forward primer, 5'-TGCTGTTTCACTGGTTATGC GG-3', and reverse primer, 5'-CCATTGCCCCTGTTTCACTATCC-3'.

Detection of Cre-mediated Recombination by beta-Galactosidase (lacZ) Staining—Cells undergoing recombination were identified using the Cre/loxP system in which the K14-Cre transgene mediated DNA recombination after being crossed with the ROSA26 reporter transgene (17). As a consequence to recombination, beta-galactosidase expression was activated and restricted to the cells where the K14 promoter was expressed at levels sufficient to cause Cre-mediated activation of the ROSA26 marker. To assess K14 promoter activity in mouse teeth, first molars were dissected from newborn mice and stained for beta-galactosidase activity. Molars were fixed over-night at 4 °C in 0.2% glutaraldehyde in phosphate-buffered saline (PBS) and washed 3 times in rinse solution (0.005% Nonidet P-40 and 0.01% sodium deoxycholate in PBS). Tissues were stained overnight at 37 °C using a staining solution containing 2 mM MgCl2, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 0.4% X-gal, rinsed twice in PBS, and post-fixed in 3.7% formaldehyde.

Cryostat Sectioning—Mandibles of newborn mice were prepared for frozen sections and stained according to standard procedures. In brief, tissues were fixed in 0.2% glutaraldehyde solution overnight at 4 °C, immersed in 10% sucrose in PBS containing 2 mM MgCl2 for 30 min at 25 °C, incubated in 30% sucrose, 50% OCT (optimum cutting temperature, Sakura Finetek USA, Inc., Torrance, CA), 2 mM MgCl2 in PBS for 1.5 h at 4 °C and embedded in OCT by freezing on dry ice. Sections were cut at a 10-µm thickness, mounted on gelatin-coated slides, fixed in 0.2% glutaraldehyde for 10 min on ice, and rinsed twice in PBS containing 2 mM MgCl2 followed by a 10-min wash in PBS with 2 mM MgCl2. Tissue sections were incubated in the detergent rinse solution (2 mM MgCl2, 0.005% Nonidet P-40, and 0.01% sodium deoxycholate in PBS) for 10 min at 4 °C, and chromogen was developed in X-gal-staining solution overnight at 37 °C in the dark.

Epithelial-Mesenchyme Separation of Molars from 3-Day Postnatal Mice—The first and second molars were dissected free of the mandible and incubated in 1% dispase (Invitrogen) in PBS for 1 h on ice. The sheets of epithelial cells were separated from the underling extracellular matrix with mesenchyme-derived cells and subjected to RNA extraction.

RNA Extraction and Reverse Transcription—Total RNA from enamel organ epithelial cells from the first and second molars of 3-day postnatal mice was extracted by using the RNA-Bee reagent (TEL-TEST, Inc., Friendswood, TX). First strand cDNA was synthesized with 100 ng of random oligodeoxynucleotide decamers using a RETROscript reverse transcription kit (Ambion, Austin, TX).

Real-time PCR Analysis—PCR was carried out with the IQ SYBR green supermix kit (Bio-Rad) in a 20-µl final volume, 3–4 mM MgCl2, and 0.2–0.4 µM each primer (final concentration). Detailed primer sequences for each target gene are shown in supplemental Table 1. For analyzing C/EBP{alpha}, amelogenin, NF-YA, and beta-actin, PCR was performed using the iCycler iQ multicolor real-time PCR detection system (Bio-Rad) for 40 cycles at 95 °C for 10 s and 55 °C for 45 s. For C/EBPbeta and C/EBP{delta} analysis, PCR was performed for 40 cycles at 95 °C for 20 s, 59 °C for 20 s, and 72 °C for 20 s. Amplification specificity was verified by analysis of the data derived from the melting curve for each sample after the manufacturer's instructions. The iCycler iQ real-time PCR detection system software Version 3.1 was used to analyze results, and the PCR base-line subtraction curve-fit function was used to determine threshold cycle (CT) values. The threshold cycle value was converted to mRNA abundance, using an algorithm provided by the manufacturer.

Electrophoresis Mobility Shift Assay—Nuclear extracts and electrophoresis mobility shift assay were performed as described previously (3).

Chromatin Immunoprecipitation (ChIP) Assays—ChIP assays were performed according to the protocol for the EZ ChIP kit (17-371, Upstate%20Biotechnology">Upstate Biotechnology, Temecula, CA). Cells were cross-linked with 1% formaldehyde for 10 min at 25 °C. After washing with ice-cold PBS, cells were lysed in SDS lysis buffer (6 x 106 cells/0.4 ml of SDS lysis buffer) for 10 min on ice and sonicated at 4 watts for 4 pulses of 10 s each using a VirSonic 60 sonicator (Virtis Co., Gardiner, NY). Cell debris were removed by centrifugation for 10 min at 13,000 x g. The soluble chromatin (100 µl per immunoprecipitation assay) was transferred to a new Eppendorf tube and diluted 10 times in ChIP dilution buffer followed by preclearing with 60 µl of protein G-agarose beads (16-201C, Upstate%20Biotechnology">Upstate Biotechnology) for 60 min at 4 °C with rotation. The precleared lysates were immunoprecipitated using antibodies for either non-immune IgG (5 µg, PP64B, Upstate%20Biotechnology">Upstate Biotechnology), C/EBP{alpha} (5 µg, sc-61x, Santa Cruz Biotechnology, Santa Cruz, CA), or C/EBP{delta} (5 µg, sc-636x, Santa Cruz Biotechnology) overnight at 4 °C with rotation. Immune complexes were collected by adding 50 µl of protein G-agarose beads and washed once with 1 ml of low salt wash buffer, once with 1 ml of high salt wash buffer, once with 1 ml of LiCl wash buffer, and twice with 1 ml of Tris-EDTA buffer. Immune complexes were eluted with elution buffer, and cross-links were reversed and treated with proteinase K. DNA was recovered using spin columns. The resulting ChIP DNA solution was diluted 100 times, and 2 µl of DNA was used for PCR detection. To amplify the 200-bp product, the primer set was 5'-GCTTCCCAAACCTATTATTGCCTG-3' and 5'-TTTCTTCCAACTCTGTGCCCC-3'; to amplify the 584-bp product, the primer set was 5'-CTACTGTAATAGTCTTGAGGTCGTGGC-3' and 5'-CGATGGTTTCTTCCAACTCT GTGC-3'.

Statistical Analysis—Statistical analysis was performed using the one-way analysis of variance test, and statistical significance is defined as p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
K14-Cre Transgenic Mice Robustly Express Cre Recombinase in the Ameloblast Cell Lineage—The K14-Cre transgenic mice were bred with R26R mice to generate transgenic animals in which expression of the beta-galactosidase occurs only upon Cremediated DNA excision. When newborn mandibles of K14-Cre;R26R mice were prepared for histologic examination, X-gal positive cells were restricted to the enamel organ of the incisor and molar teeth, including the ameloblasts (Fig. 1C). The first molar of newborn pups, with the genotype of K14-Cre;R26R, was isolated and subject to whole-mount X-gal staining. As expected, the lacZ expression was uniformly distributed in enamel organ epithelial cells, whereas no lacZ stain was observed in mesenchymal cells (Fig. 1, B, panels a– c, and C, panel f). In the lower incisor, the lacZ activity was detected only in enamel organ epithelial cells from the earliest stage of ameloblast differentiation within the stem cell compartment continuously through the fully differentiated ameloblasts lining the incisor tip region (Fig. 1C). The enamel organ of the lingual surface of the incisor, an area that does not develop as secretory ameloblasts, was also identified as lacZ-positive (Fig. 1C, open arrows). When examined in histologic sections, some unstained ameloblast cells were observed sporadically, suggesting an incomplete recombination event in some cells from the enamel organ (Fig. 1C, panel d and e, solid arrows).

K14-Cre-mediated C/EBP{alpha} Ablation in Mouse Ameloblast Cell Lineage—To specifically disrupt C/EBP{alpha} gene expression in the ameloblast cell lineage (Fig. 1A), C/EBP{alpha}fl/fl (fl, flanked by loxP sites) mice were bred with K14-Cre transgenic mice to generate C/EBP{alpha}+/– heterozygous conditional knock-out mice. Subsequent mating between C/EBP{alpha}+/– mice produced homozygous conditional knock-out mice, C/EBP{alpha}–/–. RNA extracted from molar enamel organ epithelial cells of 3-day postnatal mice was reverse-transcribed, and the first strand cDNA was subject to real-time PCR analysis.

C/EBP{alpha} mRNA transcripts were reduced significantly upon removal of one C/EBP{alpha} allele, and upon removal of the second C/EBP{alpha} allele (p < 0.01, Fig. 2A), this finding suggested successful K14-Cre-mediated C/EBP{alpha} gene excision to generate a conditional C/EBP{alpha} knock-out in the enamel organ epithelium. The level of C/EBP{alpha} in C/EBP{alpha}+/– mice was reduced to 41–55% that of levels compared with expression levels observed in wild-type control mice, whereas the level of C/EBP{alpha} in C/EBP{alpha}–/– mice was reduced to 2–32% that in wild-type control mice (Fig. 2A). C/EBP{alpha} has been demonstrated as the strong transactivator of the amelogenin gene (1, 2, 5), and ablation of the C/EBP{alpha} gene is expected to affect amelogenin expression. Therefore, we examined the expression level of amelogenin mRNA from each of these C/EBP{alpha} conditional knock-out mice that varied only by the C/EBP{alpha} allele number. Amelogenin was reduced upon removal of one C/EBP{alpha} allele, with 61–85% of the amelogenin transcript abundance in C/EBP{alpha}+/– mice compared with the abundance found in wildtype control mice (p < 0.05, Fig. 2E). Surprisingly, once two C/EBP{alpha} alleles were excised, we identified that the amelogenin mRNA transcript abundance for the C/EBP{alpha}–/– mice returned to the wild-type level (p > 0.05, Fig. 2E). This finding suggests that amelogenin transcript abundance in vivo is independent of C/EBP{alpha} expression levels. Rather, amelogenin transcript abundance is compensated by an unknown mechanism(s) upon excision of two C/EBP{alpha} alleles.


Figure 1
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  		FIGURE 1.
Analysis of Cre-mediated recombination. A, solid triangles represent the loxP sites, and E represents the EcoRI restriction endonuclease site. Primers (arrows) F7 and B4 amplified the 600-bp wild-type allele; primers F6 and B6 amplified the 400-bp C/EBP{alpha}fl/fl allele; primers F4 and B4 amplified the 900-bp C/EBP{alpha}–/– allele. B, K14-Cre mice were mated with R26R transgenic mice, and the first molars were recovered by microdissection and subjected tobeta-galactosidase (lacZ) staining. Only epithelial cells (E) of the enamel organ, including the lingual enamel organ epithelia (open arrows) show positive reaction for lacZ staining, whereas mesenchymal cells (M) are completely free of lacZ staining. C, mandibles of newborn mice were used to produce frozen sections and subjected to lacZ staining to ascertain Cre recombination on a cell-by-cell basis. Occasional unstained ameloblast cells (arrow) were observed in the maturing region (d) and secretory region (e) of the incisor and in molars (f). M1, first molar; M2, second molar.

 


Figure 2
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  		FIGURE 2.
Real-time PCR analysis. The mRNA expression level for C/EBP{alpha} (A), C/EBPbeta (B), C/EBP{delta} (C), NF-YA (D), and amelogenin (E) among wild-type or C/EBP{alpha} conditional knock-out mice was determined by real-time PCR. *, p < 0.05; **, p < 0.01. Shown in parentheses is the mRNA abundance calculated from the threshold cycle (Ct), shown as a percent of wild type (wt).

 


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

 
Several studies have suggested potential roles of C/EBPbeta and NF-Y in facilitating C/EBP{alpha}-mediated transactivation (5, 18). Functional assays were used to show that either C/EBPbeta or NF-Y alone has only marginal effects on the amelogenin promoter (Fig. 3, lane 2 and 3). Although NF-Y and C/EBPbeta enhance the amelogenin promoter some 3.5-fold (Fig. 3, lane 5), this gain is ablated in the presence of a mutated NF-YA (NF-Yam29) (Fig. 3, lane 6).

Nonetheless, it is possible that increased levels of C/EBPbeta and/or NF-Y mRNA level could lead to increased protein levels sufficient to activate the amelogenin promoter and produce enough amelogenin proteins to compensate for the loss of both C/EBP{alpha} alleles. Based upon this hypothesis, the level of C/EBPbeta and NF-YA transcripts were ascertained in this study. We did not observe a significant change in the level of C/EBPbeta among mice with either one or both C/EBP{alpha} alleles removed (p > 0.05, Fig. 2B). The expression level of C/EBPbeta in mice in which there was only one C/EBP{alpha} allele (C/EBP{alpha}+/–) was 75–134% that observed in wild-type control mice, and the expression level of C/EBPbeta in mice with no C/EBP{alpha} allele (C/EBP{alpha}–/–) was 100–189% that observed in wild-type control mice (Fig. 2B). In addition there was no significant difference in the level of NF-YA in either instance in which only the C/EBP{alpha} allele number varied (p > 0.05, Fig. 2D). The level of NF-YA in heterozygous mice (C/EBP{alpha}+/–) was 70–101% that of the expression levels observed in wild-type control mice. The level of NF-YA in conditional knock-out mice (C/EBP{alpha}–/–) was 55–125% that observed in wild-type control mice.

Standard histologic examination of the hematoxylin and eosin-stained 3-day postnatal mouse mandibles showed that the enamel thickness of incisors (supplemental Fig. S1A) and molars (supplemental Fig. S1B) was not grossly affected in mice with a complete absence of C/EBP{alpha} (C/EBP{alpha}–/–) when compared with enamel thickness of wild-type control mice. The absence of detectable diminution of the enamel thickness in the C/EBP{alpha} conditional knock-out animal is not surprising given the observed compensation that restored amelogenin mRNA levels to wild-type levels (Fig. 2E). However, it is unlikely that C/EBPbeta and NF-Y, even working in combination, are sufficient to restore amelogenin abundance to the near wild-type levels that are observed (Fig. 2) especially in the absence of significant increases in C/EBPbeta and NF-Y mRNA abundance in the teeth from C/EBP{alpha} conditional knock-out mice.

C/EBP{delta} Is Able to Activate the Mouse Amelogenin Promoter— The finding that the amelogenin mRNA level is restored upon excision of both CEBP{alpha} alleles prompted us to search for an alternative pathway in the regulation of the amelogenin gene. C/EBP{delta} was regarded as a candidate because both C/EBP{delta} and C/EBP{alpha} have similar nucleotide sequence preferences for promoter binding (19). To test of whether C/EBP{delta} could function as a transactivator of the mouse amelogenin promoter, a C/EBP{delta} expression vector was co-transfected into LS8 cells with different amelogenin promoter reporter constructs: p2207-luc (the full-length amelogenin promoter), p70-luc (the minimal amelogenin promoter), mC/EBP-p2207-luc (the full-length amelogenin promoter with the mutated C/EBP site), and mC/EBP-p70-luc (the minimal amelogenin promoter with the mutated C/EBP site). Mutation of the C/EBP site abolished the basal promoter activity of mutant reporter constructs (mC/EBP-p2207-luc and mC/EBP-p70-luc) and their C/EBP{delta}-mediated transaction, whereas the reporter gene activity of wild-type constructs (p2207-luc, and p70-luc) was increased dramatically by co-transfection with C/EBP{delta} (Fig. 4A). This level of transactivation for the amelogenin promoter by C/EBP{delta} is similar to that observed for C/EBP{alpha} (5). Taken together, these data indicate that the C/EBP site is required to maintain basal amelogenin promoter activity and is responsive to either C/EBP{delta} or C/EBP{alpha}.

Previous data has shown that NF-Y and C/EBP{alpha} synergistically activates the mouse amelogenin promoter (3). To investigate whether C/EBP{delta} has similar potency to that of C/EBP{alpha}, NF-Y and C/EBP{delta} expression plasmids were co-transfected with the p70-luc reporter construct into LS8 cells. As shown in Fig. 4B, C/EBP{delta} alone increased the promoter activity about 8-fold (lane 2), whereas exogenous expression of NF-Y in isolation had only marginal effects on the promoter (lane 3). Co-transfection of C/EBP{delta} with NF-Y served to synergistically increase the promoter activity to 16-fold (Fig. 4B, lane 5), a level that was two times more than that of C/EBP{delta} only. Furthermore, the presence of mutant NF-YA (NF-YAm29) greatly reduced the promoter activity either in the absence (Fig. 4B, lane 4) or in the presence (lane 6) of exogenous C/EBP{delta} expression. These observations demonstrate that NF-Y facilitates C/EBP{delta} similarly to the way it does for C/EBP{alpha}, to synergistically activate the mouse amelogenin promoter. However, NF-Y by itself exhibits only a marginal effect on the amelogenin promoter.

Nuclear extracts prepared from LS8 cells transfected with the C/EBP{delta} expression plasmid (NE/{delta}) were supershifted by the C/EBP{delta} antibody (Fig. 5, lane 4) as well as by the NF-Y antibody (Fig. 5, lane 6). The ChIP assay was performed to assess the interaction of the mouse amelogenin promoter with its transcription factor C/EBP{alpha} or C/EBP{delta} in ameloblast-like LS8 cells transfected with the full-length mouse amelogenin promoter containing plasmid together with either the C/EBP{alpha} or the C/EBP{delta} expression vector. As a negative control, nonimmune IgG did not show the enrichment for the amelogenin proximal promoter region, whereas both C/EBP{alpha} and C/EBP{delta} antibodies were able to enrich the promoter fragment containing the C/EBP consensus binding motif (Fig. 6). Moreover, the capacity of the endogenous amelogenin promoter to respond to C/EBP{alpha} or C/EBP{delta} alone and in the presence of NF-Y was assessed in ameloblast-like LS8 cells. The endogenous amelogenin promoter is responsive to either C/EBP{alpha} or C/EBP{delta} alone (Fig. 7, lanes 2 and 3, respectively) with each factor individually enhancing amelogenin mRNA greater than 10-fold. In the presence of NF-Y, either C/EBP{alpha} or C/EBP{delta} amelogenin mRNA transcript abundance nearly doubled (Fig. 7, lanes 5 and 6, respectively).


Figure 4
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  		FIGURE 4.
In vitro analysis of the amelogenin promoter. A, the C/EBP site is required for maintaining the basal amelogenin promoter activity and C/EBP{delta}-mediated transactivation. Various reporter constructs (p2207-luc, p70-luc, mC/EBP-p2207-luc, and mC/EBP-p70-luc), each used at 250 ng, 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 the normalization of luciferase activity with beta-galactosidase activity. The mean ± S.D. from at least three independent experiments was represented, and the level of p70-luc in the absence of exogenous C/EBP{delta} was set arbitrarily as 1. B, C/EBP{delta} and NF-Y synergize 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 "expression" vector (lane 1), C/EBP{delta} (lane 2), NF-Y (lane 3), dominant negative form of NF-Y, NF-YAm 29 (lane 4), C/EBP{delta} and NF-Y (lane 5), and C/EBP{delta} 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 reflected the mean ± S.D. of three independent experiments, with the response level of p70-luc in the absence of exogenous C/EBP{delta} set arbitrarily as 1.

 


Figure 5
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  		FIGURE 5.
Electrophoresis mobility shift assay of the C/EBP{delta} binding site. Nuclear extracts were prepared from LS8 cells transfected with the C/EBP{delta} expression plasmid (NE/{delta}). Extracts were incubated with 32P-labeled probe followed by electrophoretic separation and visualized by autoradiography. For competition assay (lane 3), 50-fold molar excesses of unlabeled probe were incubated with 32P-labeled probe. For supershift assay, extracts were preincubated with a C/EBP{delta} antibody (lane 4, sc-636x, Santa Cruz Biotechnology), normal rabbit serum (lane 5), NF-YA antibody (lane 6, sc-636x, Santa Cruz Biotechnology), and normal goat serum (lane 7). The supershifted band is denoted by the asterisks.

 
In addition, immunohistochemistry detection of C/EBP{delta} protein signals were localized within nuclei of the ameloblast cell lineage in teeth (supplemental Fig. S2) which further suggests a role for C/EBP{delta} in the regulation of amelogenin gene expression in vivo. Thus, it is reasonable to propose a hypothesis that the synergism between NF-Y and C/EBP{delta} compensates for the absence of C/EBP{alpha}, restoring amelogenin expression levels to approximately wild-type status in vivo.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Data from several in vitro experimental strategies have demonstrated that C/EBP{alpha} is a strong transactivator for amelogenin gene expression (1–3,5); however, no in vivo observation of the role for C/EBP{alpha} in amelogenin expression has been reported. As a step in exploring the function of C/EBP{alpha} in regulating amelogenin activation in vivo and to circumvent the lethal neonatal phenotype in the conventional C/EBP{alpha} knock-out mice (6, 7, 20), we generated a conditional knock-out mouse strain in which the C/EBP{alpha} allele(s) was removed upon the expression of Cre recombinase under the control of K14 promoter. We used the R26R mouse to report on the cell-specific expression of Cre recombinase. The K14 promoter drove Cre expression robustly as demonstrated by the lacZ staining in the enamel organ epithelia, specifically localizing to the ameloblast cell lineage from molars and incisors. However, some unstained ameloblast cells were observed sporadically in the lower incisor. One explanation for this "sparing" is that expression of Cre recombinase in those cells unmarked by lacZ is below the threshold required for recombination and results in an incomplete gene excision indicated by the failure to express lacZ. To prevent potential contamination from mesenchymal cells, in which K14-Cre would not be active, molars of 3-day postnatal mice were dissected, and the epithelial cells were mechanically separated from the mesenchyme.


Figure 6
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  		FIGURE 6.
ChIP analysis for the binding of C/EBP{alpha} or C/EBP{delta} on the mouse amelogenin promoter. A–C, LS8 cells (100 mm plate) were transfected with p2207-luc (500 ng) with the expression vector for either C/EBP{alpha} (800 ng) or C/EBP{delta} (800 ng) and incubated for 24 h. Cells were fixed, sonicated, and incubated with an non-immune IgG or an anti-C/EBP{alpha} antibody (sc-61x, Santa Cruz Biotechnology) or an anti-C/EBP{delta} antibody (sc-636x, Santa Cruz Biotechnology) and immunoprecipitated with protein G-agarose beads (16–201C, Upstate%20Biotechnology">Upstate Biotechnology).

 


Figure 7
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  		FIGURE 7.
NF-Y facilitates C/EBP{alpha} or C/EBP{delta} and synergistically induces the endogenous amelogenin promoter as measured by mRNA transcripts. LS8 cells were transiently transfected with 200 ng of empty vector (lane 1), expression vector for C/EBP{alpha} (lane 2), C/EBP{delta} (lane 3), NF-Y (lane 4), C/EBP{alpha} and NF-Y (lane 5), and C/EBP{delta} and NF-Y (lane 6). The mRNA expression level from the endogenous amelogenin gene was determined by real-time PCR analysis. Data represents three independent experiments, each done in triplicate. The level of amelogenin mRNA from cells treated with the empty vector was set arbitrarily as 1.

 
We examined the expression level of amelogenin with regard to the C/EBP{alpha} allele number in these conditional knock-out mice. The amelogenin mRNA transcripts decreased in the C/EBP{alpha} heterozygous conditional knock-out mice (p < 0.05, Fig. 2E). Surprisingly, in the C/EBP{alpha} homozygous conditional knock-out mice, amelogenin abundance was measured essentially at the wild-type expression level (Fig. 2E). This finding strongly implies the existence of an alternative pathway for the activation of the amelogenin gene in vivo.

We examined the level of C/EBPbeta and NF-YA in these C/EBP{alpha} conditional knock-out mice. Neither the level of C/EBPbeta nor NF-YA was significantly affected in the mice with reduced C/EBP{alpha} allele numbers and consequently reduced C/EBP{alpha} mRNA levels. This observation is correlated with the finding that C/EBPbeta and NF-Y exert only arithmetic accumulation effects in activating the amelogenin gene (Fig. 3), not the synergistic effect on the amelogenin gene as observed by over-expressing C/EBP{alpha} and NF-Y (3). Therefore, C/EBPbeta and NF-Y are not likely to be capable of activating the amelogenin gene to the wild-type level in C/EBP{alpha} conditional knock-out mice. Hematoxylin- and eosin-stained 3-day postnatal mouse mandibles failed to show that enamel thickness was dramatically affected in C/EBP{alpha} conditional knock-out mice compared with the enamel thickness observed in wild-type control mice (supplemental Fig. S1).

The amelogenin expression level in the C/EBP{alpha} conditional knock-out mice suggested an alternative mechanism that circumvents the C/EBP{alpha}-mediated activation of amelogenin expression observed in vitro (1–3, 5). In searching for the potential candidate, C/EBP{delta} was analyzed because C/EBP{alpha} and C/EBP{delta} share the same binding site to the promoter (19). Data demonstrate that C/EBP{delta}, like C/EBP{alpha}, is able to bind to the amelogenin promoter (Figs. 5 and 6) and activates the mouse amelogenin promoter (Fig. 4). NF-Y could facilitate either C/EBP{alpha} or C/EBP{delta} to induce endogenous amelogenin expression in LS8 cells (Fig. 7). Immunohistochemistry for C/EBP{delta} protein revealed a nuclear localization in ameloblast cells (supplemental Fig. S2). However, we did not observe an increased C/EBP{delta} mRNA level in the enamel organ epithelia of the C/EBP{alpha} conditional knock-out mice. One explanation is that the mRNA level of a certain gene is not always proportional to its protein levels or to the functional activity of the protein. Although there is no significant change at the mRNA level of C/EBP{delta}, the activity of C/EBP{delta} protein may be increased, due to post-transcriptional and/or post-translational modifications, an outcome that is sufficient to produce enough amelogenin proteins in the C/EBP{alpha} conditional knock-out mice to build an apparently normal enamel matrix (supplemental Fig. S1).

Several studies have demonstrated a functional redundancy in the C/EBP family (21, 22). Taken together, these data suggest that C/EBP{delta} and C/EBP{alpha} may have the functional redundancy in the regulation of mouse amelogenin gene.

Here, we report on the generation of C/EBP{alpha} conditional knock-out mice, in which C/EBP{alpha} alleles are specifically excised by Cre recombinase driven by the K14 promoter. Real-time PCR analysis demonstrated successful Cre-mediated C/EBP{alpha} ablation in mouse ameloblast cell lineages in vivo. Amelogenin mRNA levels were decreased in C/EBP{alpha} heterozygous conditional knock-out mice. However, amelogenin mRNA levels returned to the wild-type level in C/EBP{alpha} homozygous conditional knock-out mice. This finding implies an existence of an alternative pathway bypassing the C/EBP{alpha} pathway and compensating for the loss of C/EBP{alpha}. In a search for other transcription factor(s) responsible for the induction of the amelogenin gene, we identified C/EBP{delta}. Like C/EBP{alpha}, C/EBP{delta} showed a similar synergistic potency with NF-Y to activate the mouse amelogenin promoter, a finding that suggests a functional redundancy between C/EBP{alpha} and C/EBP{delta}. We have successfully utilized ChIP assay to demonstrate the binding of C/EBP{alpha} or C/EBP{delta} on the amelogenin promoter in LS8 cells in vitro. We are in the process of performing in vivo ChIP assay to investigate interactions of C/EBP{alpha} and C/EBP{delta} on the amelogenin promoter during the process of amelogenesis. The ultimate test will be to examine amelogenin expression and subsequent enamel biomineralization in C/EBP{alpha} and C/EBP{delta} double knock-out mice.


   FOOTNOTES
 
* This work was supported by National Institute for Dental and Craniofacial Research, 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 Table 1 and Figs. S1 and S2. Back

1 To whom correspondence should be addressed: The Center for Craniofacial Molecular Biology, University of Southern California, CSA 142, 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; K14, keratin 14; R26R, ROSA26 reporter; PBS, phosphate-buffered saline; X-gal, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside; ChIP, chromatin immunoprecipitation. Back


   ACKNOWLEDGMENTS
 
We thank our colleagues at the University of Southern California, Center for Craniofacial Molecular Biology and Institute for Genetic Medicine for support. We thank Dr. Sigal Gery (Cedars-Sinai Medical Center) for the C/EBP{delta} expression vector, Dr. Pierre Chambon (Institut de Genetique et de Biologie Moleculaire et Cellulaire, France) for the K14-Cre mice, Dr. Philippe Soriano (Fred Hutchinson Cancer Research Center) for the R26R mice, and Dr. Michael Wu (Millipore) for the technique support for the ChIP assay. We thank Dr. Baruch Frenkel (University of Southern California-Institute for Genetic Medicine) for help in transferring the ChIP assay to our laboratory and Dr. Henry Sucov (University of Southern California-Institute for Genetic Medicine) for critical discussion during the execution of this work. We thank the two anonymous reviewers for constructive criticisms.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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