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Enhancers Located within Two Introns of the Vitamin D Receptor Gene Mediate Transcriptional Autoregulation by 1,25-Dihydroxyvitamin D₃

Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706

Address all correspondence and requests for reprints to: J. Wesley Pike, Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin 53706. E-mail: pike{at}biochem.wisc.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
The biological actions of 1,25-(OH)₂D₃ are mediated by the vitamin D receptor (VDR), a protein that binds to target genes and alters their expression. 1,25-(OH)₂D₃ is also capable of inducing transcription of the VDR gene itself. In the present study, we explored both the capacity of 1,25-(OH)₂D₃ to induce VDR gene expression in bone cells and the mechanism instrumental to this up-regulation. After establishing the ability of 1,25-(OH)₂D₃ to stimulate VDR mRNA up-regulation both in bone in vivo and in osteoblastic cells, we screened the mouse VDR gene locus from 20 kb upstream of the gene’s transcriptional start site (TSS) to 10 kb downstream of the final exon to identify VDR binding sites using chromatin immunoprecipitation-DNA microarray (ChIP-chip) analysis. Three conserved regions were identified 20, 27, and 29 kb downstream of the TSS. VDR binding to these sites in response to 1,25-(OH)₂D₃ was confirmed by ChIP analysis and was accompanied by differential localization of retinoid X receptor, histone acetylation, and RNA polymerase II recruitment. One of these regions was able to confer 1,25-(OH)₂D₃ regulation to downstream promoters, thereby permitting identification and characterization of the regulatory element located within. Importantly, a highly conserved region within the human VDR gene analogous to that discovered in the mouse was also capable of mediating 1,25-(OH)₂D₃ response. Our results demonstrate that 1,25-(OH)₂D₃ and its receptor autoregulate the expression of the VDR gene. The location of these regulatory regions and their apparent distances from the TSS are consistent with new findings suggesting the emerging relevance of distant enhancers.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
THE VITAMIN D RECEPTOR (VDR) is a member of a large superfamily of genes whose transcription factor products function to regulate the expression of target genes involved in development, metabolism, homeostasis, and reproduction. Many of these receptors are regulated by small endocrine ligands that include the steroid hormones, vitamin D₃, and the thyroid and retinoid hormones (1, 2, 3). The VDR in particular mediates the transcriptional actions of 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃], the hormonally active form of vitamin D₃. These actions at the level of target genes in the intestine, kidney, and bone function to regulate vertebrate calcium and phosphorus homeostasis and also to ensure proper mineralization and remodeling of the adult skeleton (4, 5). During 1,25-(OH)₂D₃-mediated transcription, the VDR forms a heterodimer with a retinoid X receptor (RXR) isoform and binds to vitamin D₃ response elements (VDREs) located in the promoter regions of target genes. This binding leads to the recruitment of additional coregulatory proteins necessary for chromatin modification and transcriptional activation (3). The successful formation of these multiprotein complexes containing VDR and RXR, histone-modifying enzymes, and regulatory components of the basal transcriptional apparatus are instrumental to changes in 1,25-(OH)₂D₃-mediated gene transcription (6, 7).

Interestingly, one of the genomic targets for 1,25-(OH)₂D₃ is the VDR gene itself, a process originally termed homologous up-regulation. This type of regulation has been observed in a variety of cell culture models including mouse 3T6 fibroblasts (8), human HL-60 cells (9), and MG-63 osteosarcoma cells (10) and has also been identified in certain vitamin D₃ target tissues in vivo (11, 12, 13, 14). Although considerable evidence exists to support the up-regulation of the VDR gene by 1,25-(OH)₂D₃, delineation of the mechanism by which this occurs has not been forthcoming. This is largely due to the complexity of VDR genes, but additionally because 1,25-(OH)₂D₃ also modulates VDR protein levels via a posttranslational mechanism. Indeed, several studies have shown that 1,25-(OH)₂D₃ can stabilize the VDR protein in cultured cells and increase the protein’s half-life independently of any transcriptional component (15, 16, 17).

In animal tissues in vivo, an additional component of increased VDR gene expression is the tissue-selective character of this regulation. In the kidney, for example, 1,25-(OH)₂D₃ stimulates a dramatic increase in VDR mRNA levels. This increase, however, is entirely dependent upon levels of dietary calcium that are sufficient to maintain a normal serum calcium concentration (13, 14). In the intestine, up-regulation of the VDR mRNA does not appear to occur. Although Strom et al. showed a 10-fold increase in VDR mRNA in the duodenum of vitamin D-deficient rats after treatment with a single physiologic dose of 1,25-(OH)₂D₃ (11), a follow-up study failed to demonstrate any change in VDR mRNA levels in the duodenum under similar dietary conditions (15). More recently, a similar study in rats also concluded that, although a decrease in basal VDR mRNA levels was observed in the intestine as a result of vitamin D deficiency, daily vitamin D supplementation was unable to restore these levels to normal (18). Separate from this apparent complexity in the kidney and intestine, however, 1,25-(OH)₂D₃ is fully capable of up-regulating VDR mRNA in tissues such as the parathyroid gland (12) and skin (18). Interestingly, the in vivo effects of 1,25-(OH)₂D₃ on VDR mRNA expression in bone, another primary vitamin D target tissue, have not been investigated despite the fact that up-regulation of this gene is most strikingly evident in bone osteoblasts in culture (10, 16). Taken together, these studies support the idea that 1,25-(OH)₂D₃ up-regulates the expression of the VDR gene in vivo, although this up-regulation is clearly tissue specific and almost certainly influenced by additional, unidentified factors.

As indicated earlier, the mechanism underlying the ability of 1,25-(OH)₂D₃ to up-regulate VDR mRNA expression remains unclear due both to the complexity of the promoter region(s) within the VDR gene and to a failure to identify functional VDREs. The mouse VDR gene is characterized by a TATA-less GC-rich region upstream of exon 1 that includes four tandem Sp1 sites and two putative CCAAT box elements (19, 20). Studies of the human VDR gene have suggested that at least two and perhaps three differentially used promoters may exist (21, 22, 23), only one of which corresponds to that found in the mouse. The second promoter in the human gene is located downstream of the first and contains both a TATA box as well as multiple Sp1 sites (GC rich) (23). This promoter is not conserved in the mouse gene, although an adjacent exon (exon 2) is present (19). With regard to regulation, the upstream promoter common to both the mouse and human genes is sensitive to protein kinase A stimulators (19, 24), whereas the downstream human promoter is responsive to 17ß-estradiol, glucocorticoids, and retinoic acid (23), all known regulators of VDR gene expression. Perhaps most importantly, however, none of the promoters in either the mouse or the human gene have been demonstrated to contain identifiable VDREs or to respond to 1,25-(OH)₂D₃.

In the present study, we explored the capacity of 1,25-(OH)₂D₃ to induce VDR gene expression in bone cells in vivo. We then used several cultured osteoblastic cell models to delineate the mechanism by which 1,25-(OH)₂D₃ promotes an up-regulation of the VDR gene. Three conserved VDR binding regions were identified within two large introns located substantial distances downstream of the gene’s transcriptional start site (TSS) using high-resolution chromatin immunoprecipitation analysis method combined with DNA microarray analysis (ChIP-chip). This binding was accompanied by differential localization of RXR, histone acetylation and RNA polymerase II (RNA pol II) recruitment. One of these regions was able to confer 1,25-(OH)₂D₃ regulation to a minimal VDR gene promoter, thus allowing us to identify and characterize the regulatory element located within. Finally, a region located within the human VDR gene with high sequence homology to that in the mouse also exhibited transcriptional activity when transfected into cultured bone cells. Our results identify a direct autoregulatory mechanism whereby 1,25-(OH)₂D₃ is able to induce the synthesis of VDR mRNA.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Organization of the Mouse and Human VDR Genes
The relative organization of the human and mouse VDR genes, located on chromosomes 12 and 15, respectively, is illustrated in Fig. 1. Both the human and mouse genes have eight coding exons, with unequivocal start codons located in exon 2 of the human gene and exon 3 of the mouse gene. Proteins of 423 amino acid in the mouse and 427 amino acids in the human are produced (25). Despite this similarity, the number of noncoding exons located 5' of the bone fide translational start sites differ significantly between the two species. Thus, whereas two noncoding exons are found in the mouse gene (exons 1 and 2), at least six are found in the human gene (exons 1a-1f). An additional difference is the presence in the human gene of at least two promoters, as documented in Fig. 1.

View larger version (22K): [in this window] [in a new window]   Fig. 1. Organization of the Mouse and Human VDR Genes A, Location (March 2005 assembly) and size of the VDR gene in both the mouse and human genome. B, Linear depiction of the structure of the mouse and human VDR (hVDR) gene where the arrows indicate exons containing either an initiation or a termination codon. Functional promoters are present upstream of exon 1 in the mouse and exons 1c and 1a in the human. Dotted lines encompass exons 3–10 in the mouse gene and exons 2–9 in the human gene that encode the mouse or human VDR protein (shown). DBD, DNA binding domain; LBD, ligand binding domain.

View larger version (23K): [in this window] [in a new window]   Fig. 2. Regulation of VDR mRNA by 1,25-(OH)2D3 in Vivo in Bone A, VDR mRNA expression levels in calvarial tissue of 1,25-(OH)2D3-treated mice. Calvarial tissue was isolated 6 h after a single injection of 1,25-(OH)2D3 (0–100 ng/g bw) and total RNA subjected to qRT-PCR using primers identified in Materials and Methods. VDR mRNA levels were normalized to ß-actin expression. Each point represents the mean ± SEM (n = 5). B, Nonlinear regression analysis of VDR mRNA expression presented in panel A. C, Cyp24a1 mRNA expression levels in calvarial tissue of mice as described in panel A. qRT-PCR was performed using primers specific to mouse Cyp24a1. Cyp24a1 mRNA levels were normalized to ß-actin expression. D, Nonlinear regression analysis of Cyp24a1 mRNA expression presented in panel C. Statistical analyses in panels A and C were carried out using one way ANOVA with Dunnett’s posttest (*, P < 0.01 in comparison to vehicle).

View larger version (67K): [in this window] [in a new window]   Fig. 3. Autoregulation of VDR mRNA by 1,25-(OH)2D3 and Its Receptor in Vitro A, Induction of VDR expression levels by 1,25-(OH)2D3 in vitro. MC3T3-E1 cells were treated for 6 h with either vehicle (NT, no treatment) or 1,25-(OH)2D3 (10–10 to 10–7 M). Total RNA was isolated and subjected to RT-PCR analysis using primers specific to mouse Cyp24a1 (30 cycles), VDR (25 cycles), or ß-actin (25 cycles). These results are typical of several similar experiments. B, Effects of VDR siRNA on 1,25-(OH)2D3-induced VDR mRNA expression levels in ST2 cells. ST2 cells were transfected with 20 nM nontargeting siRNA, cyclophilin B siRNA, or mVDR siRNA. After 48 h, the cells were treated for an additional 6 h with either vehicle or 1,25-(OH)2D3 (10–7 M). Total RNA was isolated and subjected to standard RT-PCR analysis using primers identified in Materials and Methods. The cycle number for amplification of each individual transcript is as follows: VDR (20 cycles), Cyp24a1 (25 cycles), OPN (15 cycles), and ß-actin (15 cycles). These results are typical of several independent experiments.

ChIP-chip Analysis of the VDR Locus Identifies VDR Binding Sites
As discussed earlier, previous attempts to define a vitamin D regulatory region in either the mouse or human VDR genes have been uniformly unsuccessful. Based upon this failure, we elected to use ChIP-chip analysis (26) to scan the entire mouse VDR gene locus as documented in Fig. 4A (the VDR gene is transcribed on the reverse strand). ST2 cells were treated in the absence or presence of 1,25-(OH)₂D₃ for 6 h and then subjected to a standard ChIP using antibodies to VDR, RXR, or a nonspecific IgG. The precipitated DNA was isolated, amplified, and the individual samples were then labeled with either Cy3 or Cy5 (see Materials and Methods). Samples to be compared were cohybridized to a high-density oligonucleotide DNA microarray that contained the tiled region from 20 kb upstream of the TSS to 10 kb downstream of the final exon at a resolution of 50 bp. A comparison of hybridization signals generated from precipitations using 1) IgG in the presence and absence of hormone, 2) VDR in the absence or presence of hormone, 3) VDR in the presence of hormone vs. input DNA and 4) RXR in the presence of hormone vs. input DNA is seen in Fig. 4B and an expansion of the peak data is seen in Fig. 4C. As highlighted in this figure, although no activity was observed outside the gene, three regions of increased VDR and RXR binding designated S1, S2, and S3 were evident within two of the VDR gene’s introns. A fourth peak of activity located proximal to the TSS was also observed, but this peak was not reproducible under each of the conditions tested. Peak finding analysis of these data indicated that binding of the VDR and RXR to the S1 region was considerably more robust than that observed at either S2 or S3. Interestingly, signals comparing RXR in the absence and presence of 1,25-(OH)₂D₃ (data not shown) were routinely weak, suggesting the possibility that a residual level of RXR might be bound to one or more of the three regions in both the absence and presence of the hormone. Regardless, these screening data suggest the location of at least three potential intronic VDR/RXR binding sites.

View larger version (43K): [in this window] [in a new window]   Fig. 4. ChIP-chip Analysis of VDR Locus Identifies VDR-Binding Regions A, Schematic diagram of the mouse VDR gene and its 10 exons. The nucleotide base pairs indicate nucleotide location on chromosome 15 (May 2004 Assembly). The reverse arrow indicates the direction of transcription on the reverse strand. B, Individual data tracks representing the enrichment ratios of Cy5 to Cy3 hybridization intensity [log (2 )] for IgG plus or minus hormone, VDR plus or minus hormone, VDR plus hormone vs. input DNA, and RXR plus hormone vs. input DNA. The nucleotide base pairs on the x-axis indicate chromosome 15 position. C, An expanded view of the S1, S2, and S3 regions for each of the four comparisons. The highlighted areas indicate the peaks of interest.

View larger version (34K): [in this window] [in a new window]   Fig. 5. ChIP Analysis Confirms VDR Binding to the Mouse VDR S1, S2, and S3 Regions A, A schematic diagram of the mouse VDR gene extending from exon 1–4. The vertical bars represent the exons that are separated by three introns whose sizes are indicated. The location of the S1-S3 regions as well as the TSS and I regions are boxed. Horizontal pairs of arrows indicate the general location and nucleotide position (relative to the TSS) of the primer pairs used in the ChIP analysis. B, ChIP analysis of 1,25-(OH)2D3-induced VDR binding to the mouse VDR gene. MC3T3-E1 cells were treated with either vehicle or 1,25-(OH)2D3 (10–7 M) for 3 h and then subjected to ChIP analysis using antibodies to either the VDR or nonspecific IgG. The DNA precipitates were recovered and subjected to 30 cycles of PCR using the primers depicted in panel A. The amplification of input DNA (0.2% of sample) is also included. VDR binding to the Cyp24a1 gene promoter was used as a positive control. C, VDR siRNA reduces VDR binding to the mouse VDR gene. MC3T3-E1 cells were transfected with 50 nM nontargeting siRNA or VDR siRNA as described in Materials and Methods. After 72 h, cells were treated for an additional 6 h with either vehicle or 1,25-(OH)2D3 (10–7 M) and then subjected to ChIP analysis as described in panel B.

View larger version (56K): [in this window] [in a new window]   Fig. 6. Sequence Homology and Location of Putative VDREs in Mouse S1, S2, and S3 Regions A, Sequence alignment and conservation analysis of mouse VDR S1, S2, and S3 regions. The UCSC genome browser was used to generate a 10-way vertebrate alignment (600–700 bp) of the mouse S1, S2, and S3 regions (March 2005 assembly). The first track corresponds to the conservation of the VDR regions across multiple species, whereas the second and third tracks depict conservation between the mouse and rat or mouse and human genes, respectively. Signal intensity corresponds to the degree of conservation. B, Identification of putative VDREs by in silico analysis of mouse VDR gene S1, S2, and S3 regions. Putative VDREs were identified using the web-based program ConSite (http://mordor.cgb.ki.se/cgi-bin/CONSITE/consite). Asterisk indicates full conservation.

View larger version (23K): [in this window] [in a new window]   Fig. 7. The Mouse VDR S1 Region Mediates Transcriptional Response to Both 1,25-(OH)2D3 and VDR A, Location and size of the mouse VDR S1, S2, and S3 regions whose activities were investigated in the context of the TK promoter. Numbers represent the 5' and 3' ends of the regions relative to the VDR gene TSS. B, Transcriptional activity of the mouse VDR gene S1, S2, and S3 regions in the presence of 1,25-(OH)2D3. ST2 cells were cotransfected with pCH110-ßgal (50 ng), pcDNA-hVDR (50 ng), and either ptk-mVDR S1-luc, ptk-mVDR S2-luc, ptk-mVDR S3-luc, or ptk-luc control vector (250 ng), treated for 24 h with either vehicle or increasing concentrations of 1,25-(OH)2D3 (10–10 M to 10–7 M) and then evaluated for both luciferase and ß-gal activity as described in Materials and Methods. Each point represents the normalized RLU (relative light unit) average ± SEM for a triplicate set of transfections. Statistical analyses were carried out using one way ANOVA with Dunnett’s posttest (*, P < 0.01 in comparison to vehicle). C, Transcriptional activity of mouse VDR gene S1 region as a function of VDR levels. MC3T3-E1 cells were cotransfected with pCH110-ßgal, increasing amounts of pcDNA-hVDR, and either ptk-mVDR S1-luc or ptk-luc control vector. Cells were treated with either vehicle or 1,25-(OH)2D3 (10–7 M) for 24 h and then evaluated for both luciferase and ß-gal activity as described in Materials and Methods. Each point represents fold induction over vehicle (calculated from the normalized RLU average ± SEM of a triplicate set of transfections).

View larger version (19K): [in this window] [in a new window]   Fig. 8. The Mouse VDR S1 Region Confers a VDR-Dependent 1,25-(OH)2D3-Inducible Response to a Mouse VDR Minimal Promoter A, Schematic diagram of pGL3 –100/+50 mVDR-luc, pGL3 –1400/+50 mVDR-luc, and pGL3 –100/+50 mVDR S1-luc reporter constructs. B, Transcriptional activity of the mouse VDR S1 region in the context of pGL3 –100/+50 mVDR. MC3T3-E1 cells were cotransfected with pCH110-ßgal, pcDNA-hVDR, and either pGL3 –100/+50 mVDR-luc, pGL3 –1400/+50 mVDR-luc, pGL3 –100/+50 mVDR S1-luc, or pGL3-luc control vector as described in Materials and Methods. Cells were treated with either vehicle or 1,25-(OH)2D3 (10–7 M) for 24 h and then evaluated for both luciferase and ß-gal activity. Each point represents the normalized relative light unit (RLU) average ± SEM for a triplicate set of transfections. C, Transcriptional activity of S1 region cotransfected with VDR siRNA. MC3T3-E1 cells were cotransfected with pCH110-ßgal, pGL3 –100/+50 mVDR-luc or pGL3 –100/+50 mVDR S1-luc reporter vector, and 50 nM of the indicated siRNA pool as described in Materials and Methods. Cells were treated and processed as in panel B. Each point represents fold induction over vehicle (calculated from the normalized RLU average ± SEM of a triplicate set of transfections).

View larger version (66K): [in this window] [in a new window]   Fig. 9. The Mouse VDR S1 Region Contains a Functional VDRE A, Diagram showing the sequence of the wild-type and two mutant versions of VDRE 1 or VDRE 2 prepared in the context of ptk-mVDR-S1-luc. Site-directed mutagenesis of each of the half-sites located in VDRE 1 or VDRE 2 was performed as described in the Materials and Methods. B, Transcriptional activity of wild-type and mutant ptk-mVDR S1-luc reporter vectors. MC3T3-E1 cells were cotransfected with pCH110-ßgal, pcDNA-hVDR, and either wild-type ptk-mVDR S1-luc vectors, mutant ptk-mVDR S1-luc vectors, or the ptk-luc control vector. Cells were treated with either vehicle or 1,25-(OH)2D3 (10–7 M) for 24 h and then evaluated for both luciferase and ß-gal activity. Each point represents the normalized RLU average ± SEM for a triplicate set of transfections. C, Bandshift analysis of VDR/RXR heterodimer binding to the specific DNA sequence derived from VDRE 1 of the mouse VDR S1 region. Labeled duplex DNA probes from the mouse osteopontin VDRE (mOPN DR3) or the sequence corresponding to S1 VDRE 1 were incubated with purified VDR and RXR in 50 mM KCl (–) or in 150 mM KCl (+) with or without 1,25-(OH)2D3 (see left panel). Unlabeled probe (1-, 10-, or 100-fold excess) was added to the incubations in a competition bandshift assay (see right panel). Complexes were resolved on 6% nondenaturing polyacrylamide gels, dried and visualized using autoradiography.

View larger version (21K): [in this window] [in a new window]   Fig. 10. The Mouse VDR S1 VDRE 1 Activates Transcription Independently in the Context of the Mouse Minimal VDR Promoter A, Diagram of pGL3 –100/+50 mVDR VDRE x1-luc and pGL3 –100/+50 mVDR VDRE x3-luc reporter constructs. B, mVDR S1 VDRE 1 mediates transcriptional response to 1,25-(OH)2D3 in either a single or multiple copy orientation. MC3T3-E1 cells were cotransfected with pCH110-ßgal, pcDNA-hVDR, and either pGL3 –100/+50 mVDR-luc, pGL3 –100/+50 mVDR S1-luc, pGL3 –100/+50 mVDR VDRE x1-luc, pGL3 –100/+50 mVDR VDRE x3-luc, or pGL3-luc control vector as described in Materials and Methods. Cells were treated with vehicle or 1,25-(OH)2D3 (10–7 M) for 24 h and then evaluated for both luciferase and ß-gal activity. Each point represents the normalized relative light unit (RLU) average ± SEM of a triplicate set of transfections. Statistical analyses were carried out using unpaired t tests (*, P < 0.05 in comparison to vehicle).

View larger version (72K): [in this window] [in a new window]   Fig. 11. VDR/RXR Heterodimer Binding to the S1 and S3 Regions Results in CBP Coactivator Recruitment, Time-Dependent Histone 4 Acetylation and RNA pol II Enhancement A, ChIP analysis of VDR and RXR binding, coactivator levels, histone acetylation, and RNA pol II levels in the VDR gene in the absence or presence of 1,25-(OH)2D3. MC3T3-E1 cells were treated with either vehicle or 1,25-(OH)2D3 (10–7 M) for 3 h and then subjected to ChIP analysis using antibodies specific to VDR, RXR, SRC-1, CBP, tetra-acetylated histone 4, RNA pol II, or an irrelevant IgG antibody. DNA precipitates were isolated and then subjected to PCR (30 cycles) using the primers depicted in Fig. 5A. Activity at the mouse Cyp24a1 promoter was included for comparison. B, ChIP analysis of time-dependent VDR binding, SRC-1 recruitment, and histone acetylation in the VDR gene after 1,25-(OH)2D3 treatment. MC3T3-E1 cells were treated with 1,25-(OH)2D3 (10–7 M) for 0, 15, 30, or 60 min and then subjected to ChIP as in panel A using antibodies to VDR, SRC-1, tetra-acetylated histone 4, and IgG. PCR analysis was carried out as described in panel A.

View larger version (20K): [in this window] [in a new window]   Fig. 12. The Human S1 Region Activates Transcription of a ptk-hVDR S1-luc Reporter in the Presence of 1,25-(OH)2D3 A, Diagram of the cloned human S1 region located in the intronic sequence between exons 2 and 3 of the human VDR gene. B, Size and nucleotide location of the human VDR S1 region. Numbers represent the 5' and 3' ends of the region relative to the human VDR gene TSS. C, Transcriptional activity of the mouse and human S1 regions in the context of the tk promoter. MC3T3-E1 cells were cotransfected with pCH110-ßgal, pcDNA-hVDR, and either ptk-mVDR S1-luc, ptk-hVDR S1-luc, or ptk-luc control vector as described in Materials and Methods. Cells were treated with either vehicle or increasing concentrations of 1,25-(OH)2D3 (10–9–10–7 M) for 24 h and then evaluated for both luciferase and ß-gal activity. Each point represents the normalized relative light unit (RLU) average ± SEM of a triplicate set of transfections. Statistical analyses were carried out using one way ANOVA with Dunnett’s posttest (*, P < 0.05; **, P < 0.01 in comparison to vehicle).

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

We coupled ChIP-chip analysis, ChIP, and traditional cloning techniques to identify regions in the VDR gene with potential enhancer properties. These techniques resulted in the identification of three regions and at least one regulatory element that were located not only within introns in the VDR gene itself but also at significant distances from the gene’s TSS. It seems likely that these regions would not have been identified without the use of the ChIP-chip approach because more traditional work has focused upon regions relatively proximal to a gene’s active promoter. The ChIP-chip DNA tiling approach to identifying regulatory regions of multiple genes within the genome appears likely to revolutionize the way in which we search for regulatory regions of protein coding genes. Indeed, this approach is currently being used to identify not only regulatory regions on specific genes or small subsets of genes, as outlined here (26, 30, 31, 32) but also to explore the existence of binding sites for specific transcription factors at high resolution across the entire genome (33, 34, 35). Perhaps more spectacularly, genome-wide ChIP-chip analysis, now more generally termed genome-wide location analysis, is being used to establish physiologically relevant, transcription factor-based gene regulatory networks in yeast and other organisms (36, 37). Emerging evidence suggests that the existence of regulatory enhancers located at large apparent distances from gene promoters may be more common than once believed and is highlighting the potential role of chromatin looping and chromatin reorganization in transcriptional regulation (38, 39, 40, 41, 42).

While identifying regulatory regions at some distance from the TSS, our approach has also raised several issues. Accordingly, although ChIP assays have revealed the localization of VDR and its partner RXR at specific sites within the gene locus and have shown that this binding in response to 1,25-(OH)₂D₃ results in local chromatin modification, at least two of these regions were unable to confer 1,25-(OH)₂D₃ response either to their own promoter or to a heterologous promoter. Although it is always possible that the receptor binding observed in the ChIP assays at S2 and S3 reflects inappropriate cross-linking, we interpret our findings to suggest that the context in which the regulatory regions are placed within an artificial plasmid strongly influences regulatory capability. This lack of context could be related to orientation relative to the downstream promoter, the presence or absence of additional transactivators or repressors capable of binding to these enhancer regions, or to an inappropriate chromatin configuration due to plasmid peculiarities. It seems reasonable to conclude that the chromatinization of a transiently transfected plasmid, to the extent that it occurs, is unlikely to be identical with that seen in the native chromosomal state. In addition, it is also possible that regulatory regions might contribute in unique ways not measurable directly via a reporter gene approach. The complete isolation of a regulatory element does not solve this problem, again because context within surrounding DNA can aid an element’s activity. The weak transcriptional activity of a single isolated element seen in this study highlights this problem. Many of the above considerations already impact the examination of natural promoters with distant regulatory elements for which hormonal regulation in vivo has been well established. Future studies will be required to resolve the apparent differences that appear to exist between the two approaches.

Despite the inability of all the regions to direct transcriptional activity, we have learned much regarding how the VDR gene is autoregulated by its own product and 1,25-(OH)₂D₃. Aside from the demonstration that the VDR gene is autoregulated and that this autoregulation is mediated via distinct regions (and at least one VDRE) located significantly downstream of the TSS, we also discovered that the binding of VDR (and RXR) to these regions was rapid and resulted in an equally rapid acetylation of histone 4. Of interest is the fact that the gene exists in a broadly acetylated state in the absence of ligand, even at sites that do not appear to contain enhancers (the I and TSS regions) and that the changes in acetylation induced by 1,25-(OH)₂D₃ appear to be generally restricted to those regions that bind the VDR. Although we have not examined the acetylation state of the VDR gene at high resolution using ChIP-chip analysis, it seems likely that the enhancement of acetylation at these enhancer regions and likely other modification may be important to subsequent changes in the rate of VDR transcription (43, 44, 45). Perhaps more interesting is the finding that RNA pol II, although it is localized strongly at the TSS in the absence of ligand, is strikingly recruited to at least two of the enhancer regions in the presence of ligand. The ability of RNA pol II to localize at sites rather distant from gene promoters is currently emerging as a relatively common occurrence. Indeed, the presence of RNA pol II at many of these sites is now known to be associated with the production of both sense and antisense RNA transcripts, perhaps of a non-protein-coding nature (46, 47, 48). The appearance of these transcripts would also suggest that the general transcriptional apparatus might be recruited to these regions as well, and recent studies using ChIP analysis suggest that this can indeed occur (27). Although the precise function of these transcriptional complexes remains unclear, it has been speculated that enhancer regions could serve as recruitment centers to increase the local concentration of transcriptional components essential to enhanced gene expression (27). If so, this raises interesting questions as to the mechanism by which these elevated levels of the transcriptional machinery at enhancers are delivered to an active promoter and how enhancers and promoters actually differ in their properties. Our discovery of VDR regulatory elements located at significant distances from the VDR promoter region and their ability to localize RNA pol II provides valuable insight into the emerging concept that gene regulation is often controlled by enhancers located at unexpected sites along the chromosome.

In conclusion, we have identified several VDR-binding sites within two introns of the mouse VDR gene. One of these is highly active transcriptionally and contains a functional VDRE. That this region is highly conserved both structurally and functionally in the human VDR gene provides significant validity to this discovery. Our ongoing studies focus upon the specific functional roles of these enhancer regions in VDR gene expression.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Reagents
General biochemicals were obtained from Fisher Scientific (Pittsburgh, PA) and Sigma Chemical Co. (St Louis, MO). 1,25-(OH)₂D₃ was obtained from Solvay (da Weesp, The Netherlands). -MEM was purchased from Mediatech (Herndon, VA) and MEM- was obtained from Invitrogen Corp. (Carlsbad, CA). Oligonucleotide primers were obtained from IDT (Coralville, IA). Anti-VDR (H-81), RXR (N197), SRC-1 (M-341), and CBP antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-acetyl H4 antibody (06-866) was obtained from Upstate (Charlottesville, VA) and anti-RNA pol II antibody (8WG16) was obtained from Berkeley Antibody Co. (Richmond, CA). Lipofectamine Plus was obtained from Invitrogen. [³²P]-deoxy (d) ATP was obtained from NEN Life Science Products, Inc. (Boston, MA).

Animal Studies
C57BL6 wild-type mice and research diets were obtained from Harlan (Indianapolis, IN). Eight-week-old mice were fed a vitamin D and calcium-deficient diet (0.8% Sr, 0.42% P) for 7 d before being dosed with 1,25-(OH)₂D₃ by ip injection. This dietary manipulation modestly reduced circulating 1,25-(OH)₂D₃ and serum calcium levels. After 6 h, the animals were killed and calvarial tissue was collected for RNA isolation (see RNA Isolation and Analysis). A preliminary study between 0 and 24 h had established peak induction at 6 h (data not shown). Experimental protocols were reviewed and approved by the Research Animal Resources Center (University of Wisconsin-Madison, Madison, WI).

Cell Culture
Mouse MC3T3-E1 and ST2 osteoblastic cells were cultured in -MEM and MEM-, respectively. Each medium was supplemented with 10% fetal bovine serum (FBS) obtained from Hyclone (Logan, UT). 1,25-(OH)₂D₃ was added in ethanol (0.1% maximum final concentration).

RNA Isolation and Analysis
Total RNA was isolated from homogenized animal tissue or cells using Triazol reagent obtained from MRC (Cincinnati, OH). The isolated RNA was reverse transcribed using the SuperScript III Ribonuclease H Reverse transcriptase kit from Invitrogen (Carlsbad, CA) and then subjected to PCR analysis using real-time (q) or standard PCR methods. The LightCycler FastStart DNA Master SYBR Green 1 kit used for qPCR was obtained from Roche (Indianapolis, IN). Primers used included those for mß-actin (forward, TGTTTGAGACCTTCAACACCC; reverse, CGTTGCCAATAGTGATGACCT); mCyp24a1 (forward, GTGCGGATTTCCTTTGTGATA; reverse, GGTAGCGTGTATTCACCCAGA); mVDR (real time) (forward, CCCATCATCCCTAGTGTGTCC; reverse, GGCTCCTTGGTTAGTGTGGTAG), and mVDR (standard PCR) (forward, TCACTGATGTCTCCAGAGCTGGGC; reverse, TGGATAGGCGGTCCTGAATGGC).

siRNA Studies
All siRNA duplexes were obtained from Dharmacon RNA Technologies (Lafayette, CO). ST2 cells were seeded into six-well plates at a concentration of 1.5 x 10⁵ cells/well and transfected approximately 24 h later using Lipofectamine PLUS in serum and antibiotic-free medium. A total of 20 nM mVDR siRNA (D-058923-01), nontargeting siRNA pool (D-001206-13), or cyclophilin B siRNA (D-001136-01) was used for transfection. After transfection, the cells were cultured in medium supplemented with 10% FBS for 48 h before they were treated with a routine concentration of 10^–7 M 1,25-(OH)₂D₃ for 6 h. RNA isolation and standard PCR analysis was carried out using the primers listed above and a primer set for mOPN (forward, CTAACTACGACCATGAGATTGGCAG; reverse, CTTTAGTTGACCTCAGAAGATGAAC).

ChIP Assay
ChIP assays were performed as previously described (7). Primer sets used for amplifying mouse VDR regions of interest included mVDR S1 (forward, CCCCTGGCACTTTATGTTTTT; reverse, AACCCTGTCATTCTCTAACTGG), mVDR S2 (forward, GACAAAAACCTTGTGGATTGGT; reverse, CTAGAAAGAACGGGCGATGTTC), mVDR S3 (forward, AGTAATAGCCCCAGGCAGAGC; reverse, AGTTCAGTCCAACAAGACGAGT), mVDR I (forward, CTGTGGAACATGGTGGCTCT; reverse, CCTGCTGTCTGGATGTAGTA), and mVDR TSS (–519 to –1) (forward, TGGGCTAATCCTAACCAGTA; reverse, CTTCCCGTGCTTAGGGCTCG). A primer set used for amplifying mouse Cyp24a1 (forward, GGTTATCTCCGGGGTGGAGT; reverse, AGTGGCCAATGAGCACGC) was included as a positive control.

Tiled Oligonucleotide Microarray Analysis
ChIP-chip analysis was carried out as described by others (26, 49, 50). In brief, DNA was isolated by specific immunoprecipitation using the ChIP methodology described above and then subjected to ligation-mediated PCR as described (51). ChIP purified DNA was blunt-ended using T₄ polymerase, ligated to linkers with the sequence 5'-GCGGTGACCCGGGAGATCTGAATTC-3' and 5'-GAATTCAGATC-3' and subjected to repeated, low cycle PCR amplification. The resulting approximately 500-bp amplicons were then labeled with Cy3 or Cy5 dyes using an indirect labeling protocol. In this method, biotinylated deoxyuridine triphosphate is first incorporated into the individual amplicons by standard procedures and the conjugated DNA labeled subsequently with either Cy5- or Cy3-conjugated streptavidin (51). Cy3- and Cy5-labeled DNA samples are then mixed in the presence of CoT-1 DNA, denatured, and cohybridized to custom oligonucleotide microarray (Nimblegen Systems Inc., Madison, WI) as described (26). The microarrays are washed extensively and scanned using a Axon 4000B scanner at the appropriate wavelengths.

Custom oligonucleotide arrays were synthesized by Nimblegen Systems, Inc. (Madison, WI). The microarray probes consisted of maskless array, in situ-synthesized 50-oligomer oligonucleotides at 2-bp intervals representing an 84-kb screen of the mouse VDR gene from 20 kb upstream of the gene’s TSS to 10 kb downstream of the final 3' noncoding exon. The tiled arrays were synthesized in duplicate in both the forward as well as reverse directions, providing four independent measurements at each site within the gene. In addition, each analysis was carried out using two independently derived ChIP DNA samples. Both the Cyp24a1 and OPN genes as well as other candidate VDR target genes (not shown) were tiled in a similar fashion. A series of comparisons were made between 1) IgG in the presence or absence of hormone, 2) VDR in the presence or absence of hormone, 3) VDR in the presence of hormone vs. input DNA, 4) RXR in the presence or absence of hormone, and 5) RXR in the presence of hormone vs. input DNA. After sample cohybridization, the logarithmic enrichment ratio of Cy5 to Cy3 hybridization intensities (log2) were plotted as a function of chromosome nucleotide position. Although all of the peaks representative of enhanced VDR or RXR binding either in the presence of 1,25-(OH)₂D₃ or as compared with input DNA were evident visually, a peak finding algorithm was used to score the relative level of binding between the three regions identified (52).

Plasmids
Full-length hVDR and hRXR were cloned into pET-29b vector obtained from Novagen (Darmstadt, Germany) and expressed with C-terminal 6xHis tags. The pCH110-ß-galactosidase reporter plasmid and the pcDNA-hVDR vector were previously described (53). ptk-mVDR S1-luc, ptk-mVDR S2-luc, and ptk-mVDR S3-luc were prepared by cloning the appropriate mouse VDR DNA fragments obtained through DNA amplification of MC3T3-E1 genomic DNA into the ptk-luc vector using the BamHI and SalI restriction sites. Mutagenesis of both VDREs located in the ptk-mVDR S1-luc reporter vector was performed using the QuikChange Mutagenesis Kit from Stratagene (San Diego, CA). pGL3 –100/+50 mVDR-luc vector was prepared by cloning the appropriate mouse VDR DNA fragment obtained through DNA amplification of MC3T3-E1 genomic DNA into the pGL3-basic vector using the HindIII and XhoI restriction sites. To prepare pGL3 –100/+50 mVDR S1-luc, the mVDR S1 region was amplified from the ptk-mVDR S1-luc vector and cloned into pGL3 –100/+50 mVDR-luc vector using the MluI and XhoI restriction sites. The same restriction sites were used for inserting phosphorylated oligonucleotide duplexes designed from VDRE 1 of the mVDR S1 region into pGL3 –100/+50 mVDR-luc vector to prepare pGL3 –100/+50 mVDR VDRE x1-luc and pGL3 –100/+50 mVDR VDRE x3-luc. ptk-hVDR S1-luc was prepared by cloning the appropriate VDR DNA fragment obtained through DNA amplification of human Caco2 genomic DNA into the ptk-luc vector using the BamHI and SalI restriction sites.

Transfection Assays
MC3T3-E1 and/or ST2 cells were seeded into 24-well plates at a concentration of 5.0 x 10⁴ cells/well in -MEM or MEM- medium containing 10% FBS and were transfected 24 h later with Lipofectamine PLUS in serum and antibiotic-free medium. Individual wells were transfected with 250 ng of a luciferase reporter vector, 50 ng of pCH110-ßgal, and 50 ng of pcDNA-hVDR (unless otherwise indicated). A total of 50 nM nontargeting pool, cyclophilin B, or VDR siRNA was also transfected where indicated. After transfection, the cells were cultured in medium supplemented with 20% FBS with or without 1,25-(OH)₂D₃. Cells were harvested 24 h after stimulation and the lysates assayed for luciferase and ß-galactosidase activities as previously described (53). Luciferase activity was normalized to ß-galactosidase activity in all case.

DNA Bandshift Analysis
Duplex oligonucleotide probes comprised of the mouse osteopontin VDRE (54) (AACAAGGTTCACGAGGTTCACGTCT) and VDRE no. 1 of the mouse VDR S1 region (GGTCAG TGTCCTCTCTAACCCCTGGCT) (underline indicates specific VDRE sequence) were end-labeled using [³²P]-dATP. Probes were incubated with 25 ng of hVDR and 12.5 ng of hRXR in 10 mM HEPES (pH 7.4), 1 mM EDTA, 5 mM MgCl₂, 10% glycerol, 0.5 mM dithiothreitol, 0.7 mM phenylmethylsulfonyl fluoride, and 50 mM (or 150 mM) KCl buffer at room temperature for 30 min. Incubations were performed in the presence or absence of 1,25-(OH)₂D₃. Protein amounts were doubled when indicated. 1, 10, or 100x molar excess unlabeled probe was added to the incubations for the competition bandshift assay. Complexes were resolved on 6% nondenaturing polyacrylamide gels, dried, and then visualized using autoradiography.

Protein Purification
Human VDR and RXR proteins were produced using the bacterial expression vectors pET-hVDR and pET-hRXR in BL21(DE3) codon Plus RIL cells obtained from Stratagene (San Diego, CA). Soluble full-length hVDR and hRXR proteins were purified to homogeneity using sequential Ni-NTA and SP-Sepharose column chromatography (53). Two forms of RXR were present.

Statistical Analyses
All values are expressed as mean ± SEM. We evaluated differences between experimental groups by using an unpaired t test or one-way ANOVA with Dunnett’s multiple comparison post test. For in vivo studies, EC₅₀ calculations were performed using nonlinear regression analysis with a sigmoidal dose response curve. All statistical calculations were performed using the GraphPad PRISM version 4 statistical software package (GraphPad Software Inc., San Diego, CA).

	ACKNOWLEDGMENTS

 
We would like to thank Miwa Yamazaki for technical assistance and the members of the Pike laboratory for helpful discussions. We would also like to thank Adam Steinberg for preparing Fig. 4.

	FOOTNOTES

 
This work was supported by National Institutes of Health Grant AR-45173 (to J.W.P.).

First Published Online February 23, 2006

Abbreviations: bw, Body weight; CBP, cAMP response element binding protein-binding protein; ChIP, chromatin immunoprecipitation; ChIP-chip, chromatin immunoprecipitation-DNA microarray; Cyp24a1, 25 hydroxyvitamin D₃-24 hydroxylase; FBS, fetal bovine serum; MOPN, mouse OPN; mVDR, mouse VDR; 1,25(OH)₂D₃, 1,25-dihydroxyvitamin D₃; OPN, osteopontin; qRT-PCR, quantitative real-time RT-PCR; RNA pol II, RNA polymerase II; RXR, retinoid X receptor; siRNA, small interfering RNAs; SRC-1, steroid receptor coactivator-1; TK, thymidine kinase; TSS, transcriptional start site; VDR, vitamin D receptor; VDRE, vitamin D₃ response element.

Received for publication January 9, 2006. Accepted for publication February 13, 2006.

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