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Endocrinology Vol. 140, No. 9 4120-4126
Copyright © 1999 by The Endocrine Society

ARTICLES

Transcriptional Repression of the Rat Osteocalcin Gene: Role of Two Intronic CCTCCT Motifs1

Ann E. Kearns2, Kiminobu Goto3, Georgia Gianakakos, Wendy Lippmann and Marie B. Demay

Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Marie B. Demay, Wellman 501, Massachusetts General Hospital, 50 Blossom Street, Boston, Massachusetts 02114. E-mail: demay{at}helix.mgh.harvard.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The regulation of osteocalcin gene transcription is complex, involving multiple positive and negative regulators. Previous studies have demonstrated that an intronic sequence, TTTCTTT (+118 to +124) is capable of mediating transcriptional repression of osteocalcin-CAT fusion genes in cells of the osteoblast lineage, by interacting with a specific nuclear protein. Further analyses of intronic sequences have identified a second silencer motif in this region. Two copies of a CCTCCT motif are present within the first intron of the rat osteocalcin gene (+106 to +111 and +135 to +140) and are capable of mediating transcriptional repression of osteocalcin-CAT fusion genes in rat osteosarcoma cells. Transient gene expression assays of wild-type and mutant osteocalcin-CAT fusion genes into ROS 17/2.8 cells demonstrate that mutagenesis of either of these CCTCCT motifs in isolation results in a 1.6-fold increase in CAT activity relative to the parent fusion gene. Moreover, a 5-fold increase in reporter gene activity is observed when both motifs are mutated together. These sequences are also capable of suppressing osteocalcin promoter activity when placed upstream to the osteocalcin promoter. Gel retardation and southwestern analyses demonstrate that the CCTCCT motifs interact with specific proteins present in nuclear extracts from ROS 17/2.8 and UMR 106 osteosarcoma cells but not COS-7 kidney cells. Mutations that abolish suppressor function of this motif markedly impair interactions with this specific nuclear protein. These data demonstrate that at least two different silencer motifs (TTTCTTT and CCTCCT) in the first intron of the rat osteocalcin gene contribute to its transcriptional repression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OSTEOCALCIN is the most abundant noncollagenous bone matrix protein and is expressed late in osteoblast differentiation. Most of the studies of osteocalcin gene regulation to date have focused on transcriptional activators, including 1,25-dihydroxyvitamin D, Cbfa1/OSF2, FGF2, and cAMP (1, 2, 3, 4). Recent investigations in mice with targeted ablation of the osteocalcin genes, however, have revealed that lack of osteocalcin is associated with increased bone density secondary to an increase in bone formation (5). These studies suggest that factors that repress osteocalcin gene transcription may, in fact, lead to increased bone formation. Transcriptional repression of the osteocalcin gene has been shown to be mediated by glucocorticoids, TNF{alpha}, YY1, and sequences within the first exon and first intron of the osteocalcin gene (6, 7, 8, 9, 10, 11). The proteins that interact with these latter elements have not yet been isolated.

Previous studies examining the silencer activity of sequences in the first intron of the rat osteocalcin gene were directed at elucidating the role of a TTTCTTT motif in transcriptional repression (11). Mutagenesis of this motif, in the context of rat osteocalcin promoter-CAT fusion genes enhanced CAT activity approximately 10-fold after transfection into ROS 17/2.8 or UMR 106 osteosarcoma cells. When an oligonucleotide containing this sequence and flanking bases (+95 to +142) was multimerized and placed upstream of a heterologous viral promoter, suppression of reporter gene expression was observed. However, mutagenesis of the TTTCTTT motif resulted in only a partial rescue of this suppression. These data suggested that other sequences present in the multimerized oligonucleotide may also mediate silencing. Of note, the TTTCTTT motif was flanked by two CCTCCT motifs (Fig. 1BGo). Because similar pyrimidine-rich sequences have been shown to contribute to the transcriptional regulation of other genes (12, 13), we examined the functional consequences of mutations in these flanking CCTCCT motifs.



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  		Figure 1. A, Schematic representation of osteocalcin fusion genes. The numbers represent the position relative to the transcription start site. "X" indicates the introduction of a substitution mutation in the CCTCCT motifs. CAT, Chloramphenicol acetyl transferase; LUC, luciferase; CTwt, an oligonucleotide containing the wild-type sequences; CTm, an oligonucleotide containing mutations in both CCTCCT motifs; ~, ligated to. B, The sequences at the 3' end of the first exon and the 5' end of intron I are indicated. The underlined bases indicate bases mutated in T1, T2 and T3 (5' to 3'). Both CCTCCT motifs were mutated in T1/T3 ABC.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of osteocalcin-CAT fusion genes
The generation of ABC (AvaI-BamHI-CAT) and of BBC (Bal 31-BamHI-CAT) have been previously described (11). Site directed mutagenesis (U.S.E. Mutagenesis Kit, Amersham Pharmacia Biotech, Piscataway, NJ) was carried out in POC-1, which includes genomic DNA containing the 5 prime regulatory region and the entire coding region of the rat osteocalcin gene (14). A unique NsiI site was substituted for the CCTCCT at +106 to +111 to generate the T1 mutant and for CCTCCT at +135 to + 140 to generate the T3 mutant. The AvaI site was blunt-ended to permit subcloning of the SacI-Ava-I (-306–+147) fragment into SacI-SmaI M13mp18 for sequencing. The sequence of the mutated fragment was confirmed, following which the SacI-SalI fragment was excised from M13mp18 and subcloned into puOCAT (14). The SacI-SacI(-1750 to -306) fragment of BBC was then inserted in its native context to generate T1ABC and T3ABC. This subcloning strategy resulted in the introduction of stop codons into all three frames between the 3' end of the osteocalcin sequences and the initiator ATG of the CAT gene. This fragment was substituted for the same region in an ABC fusion gene that had not been subjected to mutagenesis to circumvent the possibility that additional mutations in other areas of the plasmid or fusion gene had occurred during the mutagenesis procedure. Both T1ABC and T3ABC were digested with NsiI and BamHI. The BamHI-Nsi I fragment of T1ABC containing the sequences from -1750 to +107 of the rat osteocalcin gene were ligated to the Nsi I-BamHI fragment of T3ABC containing the sequences from +140 to +147 of the rat osteocalcin gene, the CAT gene, and pUC18 to generate -(+108 to +139)ABC. This fusion gene was digested with NsiI, and a double-stranded oligonucleotide was inserted to recreate the T1ABC and T3ABC mutations with the intervening wild-type sequences (T1/T3ABC). Orientation and base composition were confirmed by DNA sequence analysis.

For experiments addressing the effects of the suppressor motifs upstream to the osteocalcin promoter, the sequences from -306 to +29 of the rat osteocalcin gene were isolated by PCR and subcloned into the SacI-XhoI sites of pGL3 basic to generate -306-OCL. Double-stranded oligonucleotides containing the wild-type and mutant DNA sequences from +101 to +142, with SacI overhangs, were subcloned into the SacI site at -306. Orientation and base composition were confirmed by DNA sequence analysis.

Cell culture and transfections
ROS 17/2.8 cells were maintained in Ham’s F-12 medium with L-glutamine supplemented with 10% FBS (Life Technologies, Inc., Gaithersburg, MD), penicillin, and streptomycin. Transfection was carried out by calcium phosphate precipitation as previously described (14). HeLa cells were grown in DMEM supplemented with 10% FBS, penicillin, and streptomycin. Tansfections were carried out using DEAE dextran. Osteocalcin-CAT fusion genes were cotransfected with Rous Sarcoma Virus (RSV)-luciferase and CAT activity was normalized for luciferase activity. For experiments addressing the effect of the suppressor sequences upstream of the osteocalcin promoter, cotransfections were performed with Renilla Luciferase under the control of the SV40 promoter and dual luciferase assays were carried out using Stop and Glo (Promega Corp., Madison WI)

Gel retardation assays
Oligonucleotides were synthesized corresponding to the sequences of interest, with GATC overhangs to permit subcloning into a BamHI site. Double-stranded oligonucleotides were labeled with {alpha} 32P-dATP by filling in recessed ends with the large fragment of DNA polymerase I. Nuclear extracts were equilibrated for 30 min at room temperature in a buffer containing 110 mM KCl, poly(dI-dC) · poly(dI-dC) (0.5 µg/µg of extract protein), with or without unlabeled competitor DNAs and then incubated with {alpha}32P-dATP labeled DNA probes for 15 min at room temperature. The protein-DNA complex was brought to 10% (vol/vol) glycerol and electrophoresed on a 4% polyacrylamide gel in 2.5% glycerol, 190 mM glycine, 1 mM EDTA, 25 mM Tris (pH 8.5) at 4 C and subjected to autoradiography after drying.

Southwestern analyses
Nuclear proteins (40 µg) were denatured, subjected to SDS-PAGE, and transferred to Hybond (Amersham Pharmacia Biotech). Prestained low molecular weight markers were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA). Following transfer of the nuclear proteins and markers, the membrane was blocked for 60 min at room temperature in Blotto (50 mM Tris-HCl pH 7.5, 50 mM NaCl, 10 mM EDTA, 5% carnation instant nonfat milk, 0.5 mM dithiothreitol). The membrane was washed in binding buffer (25 mM HEPES, pH 7.9, 25 mM NaCl, 5 mM MgCl2, 0.5 mM dithiothreitol) three times over a period of 15 min, at room temperature. The membrane was hybridized in binding buffer supplemented with 5 µg/ml denatured salmon sperm DNA and radiolabeled oligonucleotide probe, for 60 min at room temperature. After 4 washes in binding buffer (over a 30 min period), the membrane was subjected to autoradiography.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To address the transcriptional role of the CCTCCT motifs in osteocalcin gene expression, site directed mutagenesis was performed to mutate each of the two sites in isolation. As shown in Fig. 1Go, the parent plasmid, containing the rat osteocalcin gene sequences from -1750 to +147 (ABC), fused to a CAT reporter was mutated at the first and second CCTCCT motifs to generate T1 and T3 ABC, respectively. Transient gene expression assays using these reporter genes were performed in ROS 17/2.8 osteosarcoma cells. As shown in Fig. 2Go, mutagenesis of the first CCTCCT motif (T1) resulted in a 1.6-fold increase in CAT activity relative to the parent fusion gene, ABC. Introduction of the same mutation into the second CCTCCT motif (T3) had identical transcriptional consequences. Because multiple copies of this element were present in the regulatory regions of other genes (13, 15, 16, 17, 18), we examined the transcriptional consequences of mutating both CCTCCT motifs in the first intron of the rat osteocalcin gene (T1/T3 ABC, Fig. 1Go). As shown in Fig. 2Go, this T1/T3 ABC mutation resulted in a 5-fold increase in CAT activity relative to the parent plasmid, ABC, suggesting that these sequences contribute significantly to transcriptional repression of the osteocalcin gene in osteoblast-like cells.



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  		Figure 2. Relative expression of osteocalcin-CAT fusion genes in ROS 17/2.8 cells. The plasmids are indicated on the x-axis. CAT activity, corrected for cotransfected RSV-luciferase activity, is indicated on the y-axis. All values are normalized to BBC activity, defined as 100%. The numbers above the bars represent the mean and SEM. All values are derived from at least three independent transfections using at least two different plasmid preparations.

 
A sequence containing part of the coding region of the first exon and the 5' end of intron I of the rat osteocalcin gene has been shown to mediate transcriptional repression in cells of the osteoblast lineage as well as in HeLa cells (10, 19). To determine whether the CCTCCT motifs mediated transcriptional repression in HeLa cells as well, these cells were transiently transfected with ABC, BBC, and T1/T3 ABC. As shown in Fig. 3Go, the sequences in the first intron of the rat osteocalcin gene are capable of mediating transcriptional repression of osteocalcin-CAT fusion genes in HeLa cells. Similar to the results observed in the osteosarcoma cells, mutagenesis of both CCTCCT motifs results in a 4.4-fold increase in reporter gene activity, relative to the parent plasmid, ABC.



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  		Figure 3. Relative expression of osteocalcin-CAT fusion genes in HeLa cells. The plasmids are indicated on the x-axis. CAT activity, corrected for cotransfected RSV-luciferase activity, is indicated on the y-axis. All values are normalized to BBC activity, defined as 100%. The numbers above the bars represent the mean and SEM. All values are derived from at least three independent transfections using at least two different plasmid preparations.

 
To address whether these DNA sequences could maintain their negative effects on osteocalcin gene transcription when placed upstream to the native osteocalcin promoter, an oligonucleotide containing the DNA sequences from +101 to +142 was ligated upstream to -306 to +29 osteocalcin-luciferase(-306–OCL) to generate CTwtF and CTwtR (Fig. 1Go). As shown in Fig. 4Go, one copy of this oligonucleotide, containing both CCTCCT motifs, was able to suppress osteocalcin promoter activity almost 2-fold in both the forward (CTwtF) and reverse (CTwtR) orientation. Mutagenesis of both CCTCCT motifs (CTmF and CTmR, Fig. 1Go) abolished the suppressive effect of these DNA sequences (Fig. 4Go).



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  		Figure 4. The CCTCCT motifs suppress transcription in a position and orientation-independent fashion in ROS 17/2.8 cells. The plasmids are indicated on the x-axis. CTwtFand CTwtR indicate plasmids where a single copy of the sequences from +101 to +142 of the osteocalcin gene are inserted upstream to the native osteocalcin promoter in the forward and reverse orientation, respectively. Plasmids containing the same sequences, but with mutations in the CCTCCT motifs are referred to as CTmF and CTmR. Firefly luciferase activity (driven by the osteocalcin promoter), corrected for cotransfected SV40-Renilla luciferase activity, is indicated on the y-axis. All values are normalized to the parent plasmid, -306-OCL (Fig. 1Go), defined as 100% activity. The numbers above the bars represent the mean and SEM. All values are derived from at least three independent transfections using at least two different plasmid preparations.

 
Gel retardation assays were performed to examine the nuclear protein-DNA interactions of the CCTCCT motifs. As shown in Fig. 5Go, the CCTCCT motif present in T1 generates specific protein-DNA complexes (arrows) using nuclear proteins isolated from ROS 17/2.8 cells. These protein-DNA complexes are competed for by excess unlabeled T1 and T3 oligonucleotides. Oligonucleotides containing mutations in the CCTCCT motifs that result in impaired transcriptional silencing (T1M and T3M) also markedly impaired competition. A more prominent, rapidly migrating complex is also generated by the wild-type probe; however, this complex is competed for by sequences containing the mutations that abolish suppression silencing (T1M and T3M). Because of the lack of correlation between DNA-protein interactions and functional activity, this complex was not thought to represent the protein of interest. To address whether the CT-rich region in the first exon of the rat osteocalcin gene that has been implicated in its silencing(+40 ACCCTCTCT +48) (10, 19) interacted with these same nuclear proteins, an oligonucleotide containing these sequences was used in competition analyses. As shown in Fig. 5Go, this T5 oligonucleotide was unable to compete for the nuclear protein-DNA complexes generated by the T1 sequence. The wild-type oligonucleotide containing the T2 (TTTCTTT) motif was likewise unable to compete for generation of the specific protein-DNA complexes (data not shown). An identical pattern was observed when an oligonucleotide containing the T3 motif was used as a radiolabeled probe. In addition, gel retardation assays performed with the T1 and T3 probes, using HeLa and UMR 106 osteosarcoma nuclear extracts resulted in an identical pattern of competition (data not shown).



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  		Figure 5. Interaction of the CCTCCT motifs with nuclear proteins from ROS 17/2.8 cells. A double-stranded oligonucleotide containing the sequences from +98 to +118 (T1) of the rat osteocalcin gene was used as a radiolabeled probe. Competition was performed with 10- and 100-fold molar excesses of unlabeled double-stranded oligonucleotides. When incubated with ROS 17/2.8 nuclear extracts, the DNA sequences which contain the T1 motif generate two specific bands (arrows) that are competed for by excess unlabeled oligonucleotide containing the same sequences (T1) as well as an oligonucleotide containing the second CCTCCT motif (T3). No competition is observed with an oligonucleotide containing a CT rich region present in exon I (T5), nor with oligonucleotides containing mutations in the CCTCCT motifs of T1 or T3 (T1M and T3M, respectively).

 
To further explore whether the nuclear protein that interacts with these DNA sequences is ubiquitously expressed, gel retardation analyses were performed using nuclear extracts from COS-7 and MLB13MYC clone 17 cells (C17). The latter cell line is capable of acquiring markers of the osteoblast phenotype, including expression of the endogenous osteocalcin gene, upon treatment with rhBMP-2 (20). No specific DNA-protein complex was generated when nuclear extracts from COS-7 (data not shown) or untreated C17 cells were used as a source of nuclear proteins (Fig. 6Go). However, when nuclear extracts from C17 cells, treated with rh-BMP2 were used, specific complexes with mobilites identical to those observed in ROS 17/2.8 cells were generated (Fig. 6Go). These data demonstrate that the nuclear protein(s) which interacts with the CCTCCT motif is not ubiquitous, and that protein-DNA interactions at this site are induced by rhBMP-2 in an in vitro model of endochondral bone formation.



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  		Figure 6. Interaction of the CCTCCT motifs with nuclear proteins from MLB13MYC clone 17 (C17)cells. A double-stranded oligonucleotide containing the sequences from +98 to +118 (T1) of the rat osteocalcin gene was used as a radiolabeled probe. Competition was performed with 10- and 100-fold molar excesses of unlabeled double-stranded oligonucleotides. When incubated with nuclear extracts from untreated C17 cells, no specific protein-DNA complexes are observed. However, when incubated with nuclear extracts from C17 cells that have acquired markers of osteoblast differentiation in response to rhBMP-2, two specific protein-DNA complexes are generated. These complexes have the same mobility and competition pattern as those generated by the ROS 17/2.8 cells.

 
Southwestern analyses were performed to further characterize the nuclear protein-DNA interactions of these sequences. As shown in Fig. 7AGo, the wild-type oligonucleotide probe, containing the sequences from +101 to +142, recognizes two proteins (closed and open arrows) with a molecular weight of approximately 90 kDa in nuclear extracts from rhBMP-2 treated C17 cells (lane 1). These complexes are not present in untreated C17 cells or in COS-7 cells(lanes 2 and 3). Only the more slowly migrating protein is present in nuclear extracts from ROS 17/2.8 and UMR 106 osteosarcoma cells (lanes 4 and 5), whereas both proteins are present in nuclear extracts from HeLa cells (lane 6). Samples run in parallel were probed with an oligonucleotide that contains mutations in both CCTCCT motifs. As shown in panel B, despite a more prolonged exposure, only the lower band (open arrow) is seen in the nuclear extracts derived from C17 cells (treated and untreated) and in HeLa cells when the mutant probe is used. The slower migrating protein (closed arrow) is not recognized by the probe with mutations in the CCTCCT motifs, suggesting that this protein may be the transcription factor which binds to the CCTCCT motifs resulting in transcriptional repression of the osteocalcin gene.



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  		Figure 7. Southwestern analyses of the DNA protein interactions of the CCTCCT motifs. Nuclear extracts from rhBMP-2 treated (lane 1) and untreated (lane 2) MLB13MYC clone 17 cells, COS-7 cells (lane 3), ROS 17/2.8 cells (lane 4), UMR 106 cells (lane 5), and HeLa cells (lane 6) were subjected to SDS-PAGE and transferred to Hybond. Membranes were probed with oligonucleotides containing wild-type sequences (A) and sequences with mutations in the CCTCCT motifs (B). The wild-type probe recognizes a 90-kDa protein (closed arrow) in nuclear extracts from rhBMP-2 treated MYB13MYC clone 17 cells, ROS 17/2.8, UMR 106 and HeLa cells (lanes 1, 4, 5, and 6), but not from untreated MLB13MYC clone 17 or COS-7 cells (lanes 2 and 3). This band is not seen using the mutant probe (B). The open arrow in both A and B points to a band present with the wild-type and mutant probes, for reference. All samples were run in parallel and the membrane probed with the mutant sequences was exposed twice as long to confirm the absence of signal from the slower migrating protein. In each case (A and B), the membrane was cut to permit prolonged exposure of the MLB13MYC clone 17 lanes (48 h vs. 18 h).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Osteocalcin is expressed late in osteoblast maturation and its expression is limited to the skeleton. This suggests that factors that regulate osteocalcin gene expression are likely to be important for the acquisition of the osteoblast phenotype. In addition, regulators of osteocalcin gene transcription may well have a more global role in the regulation of gene expression in the mature osteoblast. Although osteocalcin is the second most abundant bone matrix protein, targeted disruption of the two osteocalcin genes in mice results in increased bone formation and decreased bone fragility as assessed by histomorphometric and biomechanical analyses (5). These data suggest that osteocalcin is an inhibitor of bone formation. By inference, therefore, transcription factors which repress osteocalcin gene expression may also inhibit the expression of other genes, which decrease bone formation (or increase resorption) and/or induce the expression of genes associated with increased bone formation. We have identified two motifs in the first intron of the rat osteocalcin gene, TTTCTTT and a repeated CCTCCT motif, which contribute to transcriptional silencing of this gene in an in vitro model of a mature osteoblast (ROS 17/2.8 cells). The silencer proteins that bind to the intronic DNA suppressor motifs are, therefore, likely to play a critical role in the maintenance of normal skeletal homeostasis. It is of interest that the TTTCTTT motif, which demonstrates strong silencing ability in its native position, is a relatively weak silencer when placed upstream, even in multiple copies (11), whereas the CCTCCT repeats are equally effective in either location.

The CCTCCT motifs we have identified as silencers of osteocalcin gene expression are the third suppressor motifs identified in the first exon and intron of the rat osteocalcin gene (10, 11). Both the TTTCTTT and the first CCTCCT suppressor motifs are preserved in mouse osteocalcin gene 2; however, the first CCTCCT motif is mutated to CCTCCG in mouse osteocalcin gene 1. The nuclear proteins that interact with the TTTCTTT (11) and CCTCCT motifs are induced in an in vitro model of endochondral bone formation, supporting the hypothesis that the silencer proteins that bind to these elements play a role in modulating osteocalcin gene transcription in the maturing osteoblast. It is of interest, however, that BMP-2 induces the expression of both the osteocalcin gene and nuclear protein binding to target DNA sequences that suppress expression of this same gene. These suppressor proteins likely act in concert with other transcriptional regulators of osteocalcin gene expression to determine the ultimate level of expression of this gene, which is thought to play an important role in skeletal homeostasis.

Both the TTTCTTT and CCTCCT motifs are also found in the regulatory region of other bone cell genes, including alkaline phosphatase (21), mouse osteopontin (22), mouse {alpha}1(I) collagen, and the gene encoding the PTH/PTHrP receptor (23). Their role in developing and mature osteoblasts may be to attenuate the effect of inducers of osteoblast gene expression, thereby contributing to the maintenance of skeletal homeostasis. It is highly improbable that the proteins that bind to these motifs repress expression of target osteoblast genes in unison. Rather, it is likely that, as in other models (24), the copy number, location and adjacent sequences modulate the pattern of expression of each gene individually, in response to transcription factor binding. Further investigations will be necessary to determine whether the relative proximity of the TTTCTTT and CCTCCT motifs to one another, in the regulatory regions of osteoblast genes, and to the transcription start site, influence their ability to mediate transcriptional silencing.

The presence of the nuclear proteins that bind the CCTCCT motif in nonosteoblastic cells suggests that they participate in the regulation of a wider range of genes. Consistent with this hypothesis, CCTCCT motifs have been implicated in the transcriptional regulation of genes expressed in a diverse range of tissues. Four copies of a CCTCCT motif are found in the 5'regulatory region of the epidermal growth factor receptor gene. Mutagenesis of the region containing these repeats, results in a decrease in transcriptional activation, suggesting that the motifs may play a role in the transcriptional regulation of this gene (12). An identical CCTCCT motif is also present in the region of the first intron of the CD4 gene that has been shown to contain negative regulatory activity required for subset-specific gene expression in T cells (25). Multiple copies of the CCTCCT motif are also present in the regulatory regions of the human prointerleukin 1ß, human {alpha}IIb intergrin, ovine LIF, and chicken ß-type globin genes (13, 16, 17, 18). Interestingly, in a fashion analogous to our current studies, the repeats in the latter gene are intronic and have been implicated in its transcriptional silencing.

As our understanding of the role of individual osteoblast proteins increases, characterization of the factors that control their regulation becomes more important. Isolation of the 90-kDa protein that interacts with the CCTCCT motifs will permit studies directed at elucidating the molecular mechanisms by which it mediates transcriptional silencing of bone cell genes, including osteocalcin. It will also permit further investigations, directed at elucidating how its interactions with other transcriptional regulators help to determine the pattern of gene expression in mature osteoblasts.


   Acknowledgments
 
We are grateful to Dr. H. M. Kronenberg for critically reviewing the manuscript and to Dr. Vicki Rosen for the MLB13MYC clone 17 cells and the rhBMP-2.


   Footnotes
 
1 This work was supported by NIH Grants DK-36597 (to M.B.D.) and an NIH Fellowship Grant (to A.E.K.). Back

2 Current address: Department of Internal Medicine and Endocrinology, Mayo Clinic, Rochester, Minnesota 55905. Back

3 Current address: Third Department of Internal Medicine, Kyushu University Faculty of Medicine, Fukuoka, Japan. Back

Received December 29, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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K. Sooy and M. B. Demay
Transcriptional Repression of the Rat Osteocalcin Gene by {delta}EF1
Endocrinology, September 1, 2002; 143(9): 3370 - 3375.
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