DNA Sequences Downstream from the Vitamin D Response Element of the Rat Osteocalcin Gene Are Required for Ligand-Dependent Transactivation

W. Bruce Sneddon, Cesar E. Bogado1, M. Susan Kiernan2 and Marie B. Demay

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The sequences in the rat osteocalcin gene that lie 3' to the vitamin D response element (VDRE) have been shown to augment transcriptional activation by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. These DNA sequences, however, are unable to bind the VDR or mediate 1,25-(OH)2D3 responsiveness independently of the VDRE. To further characterize this region, the functional properties of a series of mutant oligonucleotides were examined in transiently transfected ROS 17/2.8 cells. When these mutant oligonucleotides were expressed upstream of the heterologous herpes simplex virus thymidine kinase promoter, the bases between -420 and -414 of the rat osteocalcin gene were identified as critical for maximal transactivation by 1,25-(OH)2D3. Furthermore, mutation of these sequences in the context of the native osteocalcin promoter and enhancer totally abolished the ability of the VDRE to mediate 1,25-(OH)2D3 responsiveness. These bases, which are essential for the 1,25-(OH)2D3 responsiveness of the rat osteocalcin gene, are also present in a similar position, relative to the VDRE, in the human osteocalcin gene. To explore whether these sequences could enhance transactivation by other inducible transcription factors, they were examined for their ability to synergize with the chick vitellogenin estrogen response element and the rat somatostatin cAMP response element. When placed upstream to the herpes simplex virus thymidine kinase promoter and transfected into ROS 17/2.8 cells, these sequences were able to enhance transcriptional responsiveness to 17ß-estradiol and forskolin, respectively, demonstrating that they also contribute to transactivation by other inducible transcription factors.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Vitamin D, through its biologically active metabolite, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], interacts with target genes by binding to a nuclear steroid hormone receptor (reviewed in Ref.1), the vitamin D receptor (VDR), which heterodimerizes with the retinoid X receptor (RXR) and binds to up-regulatory vitamin D response elements (VDRE) on target genes (2, 3). As a consequence, presumably through stabilization of the transcription preinitiation complex, transcriptional activation occurs.

The VDRE of the rat osteocalcin gene has been characterized as an imperfect direct hexameric repeat separated by three bases (GGGTGAATGAGGACA) (4). Initial studies directed at characterizing this VDRE revealed that the inclusion of bases 3' to the VDRE resulted in a 3-fold increase in 1,25-(OH)2D3 responsiveness in transient gene expression assays (5). Interactions between VDREs and binding sites for other transcription factors have previously been reported. When placed upstream to the minimal adenovirus E1b promoter, the mouse osteopontin VDRE is able to synergize with SP-1, nuclear factor-1, octamer binding protein-1, and activating protein-1 binding sites (6) to augment the transcriptional response to 1,25-(OH)2D3. Although none of these binding sites is found in close proximity 3' to any of the known, naturally occurring VDREs (5, 7, 8, 9, 10, 11, 12), this observation supports the hypothesis that the level of 1,25-(OH)2D3 responsiveness of the rat osteocalcin gene may be affected by transcription factors that interact with sequences flanking the VDRE.

The classical model for steroid-activated transcription is the hormone-bound receptor binding to its response element, leading to transactivation. Recent information indicates that this process is much more complex (13, 14, 15, 16, 17). Gene promoters are thought to integrate multiple signals to modulate transcriptional responses. We have identified a sequence 3' to the VDRE of the rat osteocalcin gene that is absolutely required for VDR-mediated transactivation of the rat osteocalcin gene, suggesting that multiple factors contribute to transcriptional activation by the VDR.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In our initial studies aimed at characterizing the VDRE of the rat osteocalcin gene, we observed that an oligonucleotide containing the DNA sequences from -458 to -403 (1D3) was able to confer 6-fold 1,25-(OH)2D3 responsiveness to the herpes simplex virus thymidine kinase (HSV-tk) promoter in transient gene expression assays in ROS 17/2.8 cells (5). Further studies revealed that the minimal VDRE was contained in the bases from -456 to -442 (4) and that the DNA sequences from -458 to -427 (10D3) were only able to confer 2-fold 1,25-(OH)2D3 responsiveness in transient gene expression assays (4). The sequences from -428 to -403 did not bind the VDR, nor could they confer 1,25-(OH)2D3 responsiveness independently of the VDRE (5), thereby excluding the possibility that they contained a second VDRE. To explore the hypothesis that this region of the osteocalcin gene contained specific DNA sequences responsible for enhanced transactivation by 1,25-(OH)2D3, a series of oligonucleotides representing 3' deletions of 1D3 was synthesized and ligated into pUTKAT3 (18). Their ability to mediate transcriptional activation by 1,25-(OH)2D3 was assessed after transfection into ROS 17/2.8 cells (Fig. 1Go). The fusion gene, 14D3-tkCAT (CAT = chloramphenicol acetyltransferase; Table 1Go), which contained seven fewer bases than 1D3-tkCAT, retained a 6.0 ± 0.3-fold induction of CAT activity in response to 10-8 M 1,25-(OH)2D3 (Fig. 1Go). In contrast, 13D3-tkCAT (Table 1Go), which contained eight fewer 3' bases than 14D3-tkCAT, conferred a 2.4 ± 0.1-fold responsiveness to 1,25-(OH)2D3 (Fig. 1Go). There was no difference in basal expression between 14D3-tkCAT and 13D3-tkCAT (data not shown). The human (h) homologs of 14D3-tkCAT and 13D3-tkCAT (Table 3Go) were also examined for their ability to mediate ligand-dependent transactivation in ROS 17/2.8 cells. h14D3-tkCAT was able to respond 2.6-fold to 1,25-(OH)2D3, whereas the responsiveness of h13D3-tkCAT was only 1.2-fold.



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Figure 1. Relative Stimulation of CAT Activity in Response to 10 nM 1,25-(OH)2D3

ROS 17/2.8 cells were transiently transfected with the indicated fusion genes. Relative stimulation of CAT activity in response to 10 nM 1,25-(OH)2D3 represents the mean ± SEM of three independent experiments using at least two different preparations of each plasmid. Each transfection was performed in triplicate and normalized for transfection efficiency with RSV-luc cotransfection.

 

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Table 1. Double-Stranded Oligonucleotides Containing the Sense Sequences Indicated, with GATC Overhangs, were subcloned into the BamHI site of pUTKAT3

 

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Table 3. Double-Stranded Oligonucleotides with the Sense Sequences Shown Were Employed in Gel Retardation Analysis (Fig 7Go)

 
Because deletion of eight bases at the 3' end of the 14D3 oligonucleotide (13D3) reduced transcriptional activation by the VDRE, a series of 14D3 oligonucleotides with mutations in the region 3' to the VDRE was tested for their ability to confer 1,25-(OH)2D3 responsiveness to the HSV-tk promoter when transfected into ROS 17/2.8 cells (Table 2Go and Fig. 2Go). All fusion genes contained one copy of the oligonucleotide in the correct orientation. Mutations in the region corresponding to -420 to -414 of the rat osteocalcin gene, like 13D3-tkCAT, resulted in a 3-fold reduction in 1,25-(OH)2D3 responsiveness in ROS 17/2.8 cells. These sequences were, therefore, critical for the increased 1,25-(OH)2D3 responsiveness observed with 14D3-tkCAT.


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Table 2. Double-Stranded Mutant 14D3 Oligonucleotides Containing the Sense Sequences Indicated, with GATC Overhangs, Were Subcloned into the BamHI Site of pUTKAT3

 


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Figure 2. The Sequences Required for the Increased 1,25-(OH)2D3 Responsiveness Are Located between -420 and -414 of the Rat Osteocalcin Gene

A series of mutant 14D3 oligonucleotides was ligated to pUTKAT3 and transiently transfected into ROS 17/2.8 cells. The mutant oligonucleotides are shown in Table 2Go. Relative stimulation of CAT activity in response to 10 nM 1,25-(OH)2D3 represents the mean ± SEM of four independent experiments using at least two different preparations of each plasmid. Each transfection was performed in triplicate and normalized for transfection efficiency with RSV-luc cotransfection.

 
To assess whether these same DNA sequences augmented transcriptional activation by 1,25-(OH)2D3 in osteosarcoma cells, which do not express the endogenous osteocalcin gene, two fusion genes were expressed in UMR-106 cells. 14D3-tkCAT and a mutant with decreased responsiveness (M5-14D3-tkCAT) were examined for their ability to confer 1,25-(OH)2D3 responsiveness to a heterologous viral promoter. When 14D3-tkCAT was expressed in UMR-106 cells (Fig. 3Go), a 2.5 ± 0.4-fold induction of CAT activity was observed in response to 10-8 M 1,25-(OH)2D3, whereas M5-14D3-tkCAT only conferred a 1.3 ± 0.2-fold response (Fig. 3Go).



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Figure 3. The Sequences between -420 and -414 of the Rat Osteocalcin Gene Augment 1,25-(OH)2D3 Responsiveness in UMR-106 Cells

UMR-106 cells were transiently transfected with plasmids containing either the wild-type 14D3 or mutant M5 oligonucleotide fused to pUTKAT3. Relative stimulation of CAT activity in response to 10 nM 1,25-(OH)2D3 represents the mean ± SEM of three independent experiments using at least two different preparations of each plasmid. Each transfection was performed in triplicate and normalized for transfection efficiency with RSV-luc cotransfection.

 
To demonstrate that the sequences that augment transcriptional responsiveness to 1,25-(OH)2D3 in the context of a heterologous viral promoter do so with the native osteocalcin promoter as well, a composite M3 and M4 mutation was introduced by site-directed mutagenesis of an osteocalcin-CAT fusion gene containing the sequences from -522 to -8 of the rat osteocalcin gene. The wild-type (OC-CAT) and the mutant plasmid (M3,M4-OC-CAT) were transfected into ROS 17/2.8 cells to examine their ability to mediate transactivation by 1,25-(OH)2D3 (Fig. 4Go). The M3-M4 composite mutation abolished 1,25-(OH)2D3 responsiveness, demonstrating that these sequences are essential for 1,25-(OH)2D3 induction of rat osteocalcin gene expression in the context of the native promoter. The combined M1-M2 mutation, which separately had no effect on the transcriptional responsiveness to 1,25-(OH)2D3 in the context of a heterologous viral promoter, also abrogated 1,25-(OH)2D3 responsiveness (Fig. 4Go), indicating that sequences 5' to the GGTTTGG are involved in transcriptional activation by 1,25-(OH)2D3. Neither the M3-M4 nor the M1-M2 mutation had an effect on the basal expression of CAT activity (data not shown).



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Figure 4. The Bases from -420 to -414 Are Essential for the 1,25-(OH)2D3 Responsiveness of the Rat Osteocalcin Gene

A composite mutation containing the base changes in M3 and M4 was introduced into the 5'-flanking region of the rat osteocalcin gene by site-directed mutagenesis to yield M3,M4-OC-CAT. The same procedure was employed to introduce a composite mutation containing the base changes in M1 and M2 to yield M1,M2-OC-CAT. ROS 17/2.8 cells were transiently transfected with the mutant and wild-type plasmids, and their ability to induce CAT activity in response to 10 nM 1,25-(OH)2D3 was assessed. Relative stimulation of CAT activity represents the mean ± SEM of three independent experiments using at least two different preparations of each plasmid. Each transfection was performed in triplicate and normalized for transfection efficiency with RSV-luc cotransfection.

 
Experiments were undertaken to address whether the sequences shown to augment 1,25-(OH)2D3 responsiveness could modulate transactivation by other transcription factors. The estrogen response element from the chick vitellogenin gene (19) was substituted for the VDRE in both 14D3-tkCAT and 9D3-tkCAT (Fig. 5Go). These fusion genes were transfected into ROS 17/2.8 cells and assayed for transcriptional induction by 10-8 M 17ß-estradiol. In cells transfected with 14ERE-tkCAT (ERE = estrogen response element), 17ß-estradiol induced CAT reporter activity 3.1 ± 0.4-fold (Fig. 5Go). In contrast, cells transfected with 9ERE-tkCAT only displayed a 1.4 ± 0.4-fold response to 17ß-estradiol (Fig. 5Go). To determine whether the augmented transcriptional responsiveness mediated by these sequences extended beyond the nuclear receptor superfamily, the cAMP response element from the rat somatostatin gene (20) was substituted for the VDRE in both 14D3-tkCAT and 9D3-tkCAT (Fig. 6Go). These fusion genes were transfected into ROS 17/2.8 cells and assayed for responsiveness to 10-6 M forskolin. In cells transfected with 14CRE-tkCAT (CRE = cAMP response element), forskolin induced CAT activity 3.3 ± 0.4-fold (Fig. 6Go), whereas 9CRE-tkCAT mediated a 1.3 ± 0.1-fold responsiveness to forskolin. These data demonstrate that the augmented transcriptional responsiveness mediated by the sequence we have identified is not limited to its interactions with a VDRE.



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Figure 5. The Sequences between -420 and -414 Augment Transcriptional Responsiveness Mediated by an ERE

The ERE from the chick vitellogenin II gene was substituted for the VDRE in the 14D3 and 9D3 oligonucleotides to generate 14-ERE and 9-ERE. Double stranded oligonucleotides containing the sense sequence shown, with GATC overhangs, were ligated into the BamHI site of pUTKAT3 and transiently transfected into ROS 17/2.8 cells. The arrows indicate the ERE. Relative stimulation of CAT activity in response to 10 nM 17ß-estradiol represents the mean ± SEM of three independent experiments using at least two different preparations of each plasmid. Each transfection was performed in triplicate and normalized for transfection efficiency with RSV-luc cotransfection.

 


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Figure 6. The Sequences between -420 and -414 Augment Transcriptional Responsiveness Mediated by a CRE

The rat somatostatin cAMP response element was substituted for the VDRE in the 14D3 and 9D3 oligonucleotides to generate 14-CRE and 9-CRE. Double stranded oligonucleotides containing the sense sequences shown, with GATC overhangs, were ligated into BamHI site of pUTKAT3 and transiently transfected into ROS 17/2.8 cells. The CRE is indicated by the line. Relative stimulation of CAT activity in response to 1 µM forskolin represents the mean ± SEM of seven independent experiments performed using at least two different preparations of each plasmid. Each transfection was performed in triplicate and normalized for transfection efficiency with RSV-luc cotransfection.

 
To examine the protein/DNA interactions in the region of the rat osteocalcin gene 3' to the VDRE, gel retardation analysis was performed. A double stranded oligonucleotide, -D14D3 (14D3 lacking the VDRE), representing the rat osteocalcin sequence between -434 and -410 was employed as a probe (Table 3Go). Gel retardation analysis was performed using nuclear extracts from ROS 17/2.8 (Fig. 7AGo) and UMR-106 (Fig. 7BGo) cells. Two major retarded bands (see arrows in Fig. 7Go) were observed, all of which were competed for by excess unlabeled -D14D3, wild-type 14D3, and the human homolog of 14D3 (h14D3). Double stranded oligonucleotide competitors, containing mutations that abolished 1,25-(OH)2D3 responsiveness in the context of the native osteocalcin promoter (-DM3,M4 and -DM1,M2’ Table 3Go), failed to compete for two high mol wt protein-DNA complexes present in nuclear extracts from either ROS 17/2.8 or UMR-106 cells (Fig. 7Go, A and B).



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Figure 7. The Sequences 3' to the Rat Osteocalcin VDRE Generate Two High Mol Wt Protein-DNA Complexes in ROS 17/2.8 (A) and UMR-106 (B) Nuclear Extracts

-D14D3 was employed as a probe (Table 3Go) in gel retardation assays. Competitor oligonucleotides (10- and 100-fold molar excess) were preincubated with nuclear extracts before probe addition, as outlined in Materials and Methods. The two high mol wt protein-DNA complexes are indicated by the arrows.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
1,25-(OH)2D3-mediated transactivation of the rat osteocalcin gene is initiated by the binding of the hormone-bound VDR/RXR heterodimer to the VDRE (2, 3, 4). The bases 3' to the VDRE have previously been shown to influence the level of 1,25-(OH)2D3 responsiveness of this gene (5). The experiments presented herein demonstrate that the bases between -420 to -414 of the rat osteocalcin gene are essential for the 1,25-(OH)2D3 responsiveness of the rat osteocalcin gene in the context of the native promoter, although this region does not independently bind the VDR, nor does it confer 1,25-(OH)2D3 responsiveness independently of the VDRE (5). Furthermore, because these DNA sequences increase 1,25-(OH)2D3 responsiveness in the context of both the native rat osteocalcin promoter and the heterologous HSV-tk promoter, the positions of these bases, with respect to the transcription preinitiation complex, are not crucial. The transcriptional activity of this region is not limited to VDRE-mediated transactivation, as we have shown that it is capable of augmenting transcription in concert with either an ERE or a CRE.

Accessory factors have been shown to contribute to transcriptional activation mediated by the estrogen, thyroid hormone, and retinoic acid receptors (13, 14, 15, 16, 17, 21, 22). Several positive cofactors have been identified, including TRIP1 (23), TIF1 (24), RIP140, RIP160 (14, 16), and SRC-1 (17). These proteins appear to mediate their positive effects on transcriptional activation through an interaction with the C-terminal transactivation domain of the steroid receptor. Although these proteins have not been shown to bind DNA, sequences adjacent to the steroid response element in question may contribute to binding or transactivation by these accessory proteins.

The mechanism by which the identified sequences in the rat osteocalcin gene enhance transcriptional responsiveness has not yet been determined. Gel retardation assays indicate that this region does not affect the affinity of the VDR/RXR heterodimer for the VDRE in either porcine intestinal (5) or ROS 17/2.8 nuclear extracts (data not shown). Gel retardation assays indicate that the sequences between -434 and -410 (3' to the rat osteocalcin VDRE) generate two protein-DNA complexes when incubated with ROS 17/2.8 and UMR-106 nuclear extracts. The identified bases may, therefore, bind a transcription factor that promotes 1,25-(OH)2D3-mediated transactivation by facilitating interactions between the receptor-occupied VDRE and the basal transcription apparatus. The VDR has been shown to interact with transcription factor IIB via its carboxyl-terminal region (25), which is also involved in receptor heterodimerization and ligand-induced transactivation (26). When bound to their respective sites, the VDR/RXR heterodimer and the factor that interacts with the sequences identified may recruit unique coactivator proteins to the transcription initiation complex or, together, efficiently recruit a single protein, resulting in synergistic transcriptional activation (27). To date, the GGTTTGG sequence present from -420 to -414 has not been identified as a transcription factor-binding site. In composite mutations, upstream sequences have also been identified as functionally relevant, suggesting that the response element extends beyond the GGTTTGG motif.

The GGTTTGG sequence that is critical for the 1,25-(OH)2D3 responsiveness of the rat osteocalcin gene is also present in the human osteocalcin gene (10), and we have shown that the region 3' to the VDRE in the human osteocalcin gene enhances ligand-dependent transactivation 2-fold. Comparison of the rat and human osteocalcin sequences immediately 3' to their respective VDREs (Table 3Go) indicates that of the eight bases identified by mutagenesis experiments (between -425 and -416 of the rat sequence) as being critical for 1,25-(OH)2D3 responsiveness in the context of the native promoter, seven are conserved. In contrast, in the region immediately 5' to the functionally important sequences, corresponding to between -434 and -426 of the rat sequence, only four of nine bases are conserved. Taken together, these data suggest that the boundaries for the identified element are likely to lie between -429 and -414 of the rat osteocalcin gene. Analysis of other 1,25-(OH)2D3- (7, 8, 9) and estrogen-responsive genes (19, 22, 28, 29, 30, 31) failed to reveal a homologous sequence. However, further definition of the exact base requirements for ligand-activated transcription of the rat osteocalcin gene may yield new and important information.

We have identified an element, distinct from the VDRE, that is required for induction of rat osteocalcin gene transcription by 1,25-(OH)2D3. Our data lend credence to the hypothesis that transactivation by steroid hormone receptors involves more than the occupied receptor binding to its response element. Characterization of accessory proteins and identification of novel enhancer elements will provide important insights into the mechanism of transcriptional activation by 1,25-(OH)2D3 and other steroid hormones.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Synthesis of CAT Fusion Genes
For experiments using a heterologous promoter, oligonucleotides were inserted into the BamHI site of pUTKAT3 (18) (a gift from Dr. David Moore, Massachusetts General Hospital, Boston, MA). Oligonucleotides were synthesized (on an Applied Biosystems model 380A synthesizer, Foster City, CA) corresponding to the sequences of interest, with the addition of 5'-bases (GATC) to permit cloning into the BamHI site of pUTKAT3. Orientation and copy number of the oligonucleotides were determined by DNA sequencing.

For experiments using the native rat osteocalcin promoter, the plasmid 522-CAT, which contains 522 bp upstream from the transcriptional start site of the rat osteocalcin gene fused to the CAT gene, was employed (4). Mutations in regions of interest were introduced by site-directed mutagenesis using the U.S.E. Mutagenesis kit (Pharmacia, Piscataway, NJ). Two oligonucleotide primers were employed in this procedure. A oligonucleotide (50-mer) that eliminated the XbaI site in the polylinker of pUC18 (CGACTCTAGA to CGACgaTAGA) was used as a selection primer. The mutation of interest was introduced into a second primer. The mismatched bases were located in the central portion of an oligonucleotide of 54 bases. The position of the mutation was between bases -420 and -416 of the rat osteocalcin gene (CCTGGGGTTTGGCTCC to CCTGGttTggGGaTCC). The conversion of C to A in position 413 created a BamHI site, which was used to screen for the introduction of the mutation. 1,25-(OH)2D3 responsiveness in cells transfected with the fusion gene that contained this base change alone in the context of the native (data not shown) or HSV-tk promoters (Fig. 2Go, M6-14D3) was the wild type. The sequences from -522 to -306 (SacI site) were sequenced to confirm the introduction of these mutations and to exclude the presence of other undesired mutations. This region was then substituted for the identical nonmutated region in the wild-type parent plasmid (OC-CAT) to yield M3,M4-OC-CAT. A similar procedure was employed to introduce a mutation in bases between -425 to -421 (CCTGGGGTTTGGCTCC to aaTttGGTTTGGaTCC) to yield M1,M2-OC-CAT.

Cell Culture and Transfections
ROS 17/2.8 cells were maintained in Ham’s F-12 medium with L-glutamine (Life Technologies, Grand Island, NY) supplemented with 10% (vol/vol) FBS, penicillin, and streptomycin. From 24 h before transfection until harvesting, cells were cultured in medium containing charcoal-stripped FBS. Transfections were performed using the calcium phosphate method, as previously described (32). UMR-106 cells were maintained in DMEM (Life Technologies) supplemented with 10% (vol/vol) FBS, penicillin, and streptomycin. From 24 h before transfection until harvesting, cells were maintained in medium containing charcoal-stripped FBS. Transfections were performed by lipofection (Life Technologies) with 10 µg test plasmid/well of a six-well plate. As with the ROS 17/2.8 cells, UMR-106 cells were treated with 10-8 M 1,25-(OH)2D3 immediately after transfection and again the following day. The cells were fed and stimulated 40 h posttransfection and harvested 24 h later. A similar time course of hormone treatment was used in the studies in which ROS 17/2.8 cells were treated with 10-8 M 17ß-estradiol and 10-6 M forskolin. For the transfection assays employing the ERE-tkCAT fusion genes, ROS 17/2.8 cells were maintained in phenol red-free Ham’s F-12 medium with L-glutamine (Life Technologies). CAT activity was assessed as previously described (33). All test plasmids were cotransfected with a control plasmid containing the luciferase gene under the control of the Rous sarcoma virus (RSV) promoter. Luciferase activity was measured using a standard protocol (34). The presence of 1,25-(OH)2D3, 17ß-estradiol, or forskolin did not affect the level of luciferase activity. CAT activity was assessed by densitometric scanning of TLC plate autoradiograms, and each replicate was normalized for luminescence units. Relative CAT activity, presented as fold stimulation, reflects the ratio of corrected CAT activity in the presence or absence of inducer.

DNA Sequencing
All DNA sequencing was carried out by the dideoxynucleotide chain termination method after subcloning into M13 vectors (35).

Gel Retardation Assays
Oligonucleotides were labeled by filling in recessed ends with the large fragment of DNA polymerase I and [{alpha}-32P]deoxy-ATP. The oligonucleotide used as a probe for these studies was the sequence 3' to the rat osteocalcin VDRE between bases -434 and -410 (-D14D3). The sequence of its sense strand and those of competitor oligonucleotides are shown in Table 3Go. Gel retardation assay buffer composition and nuclear extract preparation were described previously (5). ROS 17/2.8 or UMR-106 cell nuclear extracts were preincubated with poly(dI-dC) at a concentration of 0.5 µg/µg extract protein along with 10 or 100 ng of each unlabeled competitor, as indicated, for 15 min at 22 C. Subsequently, 1 ng probe was added for an additional 15 min. The mixture was brought to 10% (vol/vol) glycerol and electrophoresed on a 4% polyacrylamide gel.


    FOOTNOTES
 
Address requests for reprints to: Marie Demay, M.D., Endocrine Unit Wellman 501, Massachusetts General Hospital, Boston, Massachusetts 02114.

This work was supported by NIH Grant DK-36597.

1 Present address: Instituto de Investigaciones Metabolicas, Buenos Aires, Argentina. Back

2 Present address: University of Vermont Medical School, Burlington, Vermont. Back

Received for publication April 10, 1996. Revision received October 28, 1996. Accepted for publication October 30, 1996.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Beato M 1989 Gene regulation by steroid hormones. Cell 56:335–344[Medline]
  2. Yu VC, Delsert C, Anderson B, Holloway JM, Devary OV, Näär AM, Kim SY, Boutin J-M, Glass CK, Rosenfeld MG 1991 RXRß: a coregulator that enhances binding of retinoic acid, thyroid hormone, and vitamin D to their cognate response elements. Cell 67:1251–1266[Medline]
  3. Kliewer SA, Umesono K, Heyman RA, Evans RM 1992 Retinoid X receptor interacts with nuclear receptors in retinoic acid, thyroid hormone, and vitamin D3 signalling. Nature 355:446–449[CrossRef][Medline]
  4. Demay MB, Kiernan MS, DeLuca HF, Kronenberg HM 1992 Characterization of 1,25-dihydroxyvitamin D3 receptor interactions with target sequences in the rat osteocalcin gene. Mol Endocrinol 6:557–562[Abstract]
  5. Demay MB, Gerardi JM, DeLuca HF, Kronenberg HM 1990 DNA sequences in the rat osteocalcin gene that bind the 1,25-dihydroxyvitamin D3 receptor and confer responsiveness to 1,25-dihydroxyvitamin D3. Proc Natl Acad Sci USA 87:369–373[Abstract]
  6. Liu M, Freedman LP 1994 Transcriptional synergism between the vitamin D3 receptor and other nonreceptor transcription factors. Mol Endocrinol 8:1593–1604[Abstract]
  7. Gill RK, Christakos S 1993 Identification of sequence elements in mouse calbindin-D28k gene that confer 1,25-dihydroxyvitamin D3- and butyrate-inducible responses. Proc Natl Acad Sci USA 90:2984–2988[Abstract]
  8. Cao X, Ross FP, Zhang L, MacDonald PN, Chappel J, Teitelbaum SL 1993 Cloning of the promoter for the avian integrin ß3 subunit gene and its regulation by 1,25-dihydroxyvitamin D3. J Biol Chem 268:27371–27380[Abstract/Free Full Text]
  9. Chen K-S, DeLuca HF 1995 Cloning of the human 1-alpha,25-dihydroxyvitamin D3 24-hydroxylase gene promoter and identification of two vitamin D-responsive elements. Biochim Biophys Acta Gene Struct Express 1263:1–9[Medline]
  10. Morrison NA, Shine J, Fragonas J-C, Verkest V, McMenemy ML, Eisman JA 1989 1,25-Dihydroxyvitamin D-responsive element and glucocorticoid repression in the osteocalcin gene. Science 246:1158–1161[Medline]
  11. Noda M, Vogel RL, Craig AM, Prahl J, DeLuca HF, Denhardt DT 1990 Identification of a DNA sequence responsible for binding of the 1,25-dihydroxyvitamin D3 receptor and 1,25-dihydroxyvitamin D3 enhancement of mouse secreted phosphoprotein 1 (Spp-1 or osteopontin) gene expression. Proc Natl Aca Sci USA 87:9995–9999[Abstract]
  12. Ducy P, Karsenty G 1995 Two distinct osteoblast-specific cis-acting elements control expression of a mouse osteocalcin gene. Mol Cell Biol 15:1858–1869[Abstract]
  13. Cavailles V, Dauvois S, Danielian PS, Parker MG 1994 Interaction of proteins with transcriptionally active estrogen receptors. Proc Natl Acad Sci USA 91:10009–10013[Abstract/Free Full Text]
  14. Halachmi S, Marden E, Martin G, MacKay H, Abbondanza C, Brown M 1994 Estrogen receptor-associated proteins-possible mediators of hormone-induced transcription. Science 264:1455–1458[Medline]
  15. Kurokawa R, Soderstrom M, Horlein A, Halachmi S, Brown M, Rosenfeld MG, Glass CK 1995 Polarity-specific activities of retinoic acid receptors determined by a co-repressor. Nature 377:451–454[CrossRef][Medline]
  16. Cavailles V, Dauvois S, L’Horset F, Lopez G, Hoare S, Kushner PJ, Parker MG 1995 Nuclear factor RIP140 modulates transcriptional activation by the estrogen receptor. EMBO J 14:3741–3751[Abstract]
  17. Oñate SA, Tsai SY, Tsai M-J, O’Malley BW 1995 Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 270:1354–1357[Abstract]
  18. Prost E, Moore DD 1986 CAT vectors for the analysis of eukaryotic promoters and enhancers. Gene 45:107–111[CrossRef][Medline]
  19. Burch JBE 1984 Identification and sequence analysis of the 5' end of the major chicken vitellogenin gene. Nucleic Acids Res 12:1117–1135[Abstract]
  20. Montminy MR, Sevarino KA, Wagner JA, Mandel G, Goodman RH 1986 Identification of the cyclic-AMP-responsive element within the rat somatostatin gene. Proc Natl Acad Sci USA 83:6682–6686[Abstract]
  21. Suen C, Chin WW 1995 A potential transcriptional adaptor(s) may be required in thyroid hormone-stimulated gene transcription in vitro. Endocrinology 136:2776–2783[Abstract]
  22. Maurer RA, Notides AC 1987 Identification of an estrogen-responsive element from the 5' flanking region of the rat prolactin gene. Mol Cell Biol 7:4247–4254[Medline]
  23. Lee JW, Ryan F, Swaffield JC, Johnston SA, Moore DD 1995 Interaction of thyroid hormone receptor with a conserved transcriptional mediator. Nature 374:91–94[CrossRef][Medline]
  24. LeDouarin B, Zechel C, Garnier JM, Lutz Y, Tora L, Pierrat B, Heery D, Gronemeyer H, Chambon P, Losson R 1995 The N-terminal part of TIF1, a putative mediator of the ligand-dependent activation function (AF-2) of nuclear receptors, is fused to B-raf in the oncogenic protein T18. EMBO J 14:2020–2033[Abstract]
  25. MacDonald PN, Sherman DR, Dowd DR, Jefcoat Jr SC, DeLisle RK 1995 The vitamin D receptor interacts with general transcription factor IIB. J Biol Chem 270:4748–4752[Abstract/Free Full Text]
  26. Whitfield GK, Hsieh J, Nakajima S, MacDonald PN, Thompson PD, Jurutka PW, Haussler CA, Haussler MR 1995 A highly conserved region in the hormone-binding domain of the human vitamin D receptor contains residues vital for heterodimerization with retinoid X receptor and for transcriptional activation. Mol Endocrinol 9:1166–1179[Abstract]
  27. Guarente L 1995 Transcriptional coactivators in yeast and beyond. Trends Biochem Sci. 20:517–521
  28. Walker P, Germond J-E, Brown-Luedi M, Givel F, Wahli W 1984 Sequence homologies in the region preceding the transcription initiation site of the liver estrogen-responsive vitellogenin and apo-VLDLII genes. Nucleic Acids Res 12:8611–8626[Abstract]
  29. Berwaer M, Monget P, Peers B, Mathy-Hartert M, Bellefroid E, Davis JRE, Belayew A, Martial JA 1991 Multihormonal regulation of the human prolactin gene expression from 5000 bp of its upstream sequence. Mol Cell Endocrinol 80:53–64[CrossRef][Medline]
  30. Shupnik MA, Weinmann CM, Notides AC, Chin WW 1989 An upstream region of the rat leutinizing hormone ß gene binds estrogen receptor and confers estrogen responsiveness. J Biol Chem 264:80–86[Abstract/Free Full Text]
  31. Hobson GM, Molloy ER, Benfield PA 1990 Identification of cis-acting regulatory elements in the promoter region of the rat brain creatine kinase gene. Mol Cell Biol 10:6533–6543[Medline]
  32. Demay MB, Roth DA, Kronenberg HM 1989 Regions of the rat osteocalcin gene which mediate the effect of 1,25-dihydroxyvitamin D3 on gene transcription. J Biol Chem 264:2279–2282[Abstract/Free Full Text]
  33. Demay MB, Kiernan MS, DeLuca HF, Kronenberg HM 1992 Sequences in the human parathyroid hormone gene that bind the 1,25-dihydroxyvitamin D3 receptor and mediate transcriptional repression in response to 1,25-dihydroxyvitamin D3. Proc Natl Acad Sci USA 89:8097–8101[Abstract]
  34. Ausubel FM, Brent R, Kingston RE, et al. 1992 Current Protocols in Molecular Biology. Greene and Wiley-Interscience, New York, pp 9.7.12–9.7.14
  35. Sanger F, Nicklen S, Coulson AR 1977 DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467[Abstract]