©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Two Vitamin D Response Elements Function in the Rat 1,25-Dihydroxyvitamin D 24-Hydroxylase Promoter (*)

(Received for publication, July 29, 1994; and in revised form, October 12, 1994)

Claudia Zierold Hisham M. Darwish Hector F. DeLuca (§)

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

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The interaction between the two vitamin D response elements (DRE) located at -154 to -134 base pairs (bp) and -262 to -238 bp from the transcription initiation site has been studied using reporter gene assays and binding assays by electrophoretic gel shift measurements. 3 half-sites separated by 3 bp were found necessary for transactivation by the -154 to -125 DRE, while 2 half-sites separated by 3 bp were needed for the DRE at -262 to -238 to function. However, the two DREs together provided maximal activity. The 93-bp fragment separating the two DREs was not required and could be deleted. The most effective binding by receptor was found with the two complete DREs (dissociation constant (K) = 13.7 pM), although each DRE bound to the receptor and nuclear accessory factor with about 5 nMK. The two DREs (a total of 5 half-sites) apparently account for most if not all of the transactivation of the rat 24-hydroxylase by 1,25-dihydroxyvitamin D(3). This system represents the most powerful of the DREs reported to date.


INTRODUCTION

Vitamin D(3) is essential for the maintenance of calcium homeostasis in higher animals through its effects on kidney, intestine, and bone(1) . In addition, vitamin D has been reported to function in cell differentiation, the immune system, and fertility(2) . These functions are mediated through 1,25-(OH)(2)D(3), (^1)the active hormonal form of the vitamin. 1,25-(OH)(2)D(3) is produced by two successive hydroxylations of vitamin D. First, it is hydroxylated in the liver at C-25 to produce 25-OH-D(3), the major circulating form of the vitamin. Subsequently, 25-OH-D(3) is further hydroxylated in the kidney by the 1alpha-hydroxylase to produce 1,25-(OH)(2)D(3)(1, 3) . Both 25-OH-D(3) and 1,25-(OH)(2)D(3) can be further hydroxylated by 24-hydroxylase to produce 24,25-(OH)(2)D(3) and 1,24,25-(OH)(3)D(3), respectively. 24-Hydroxylation is believed to be the first step in the degradative pathway of vitamin D(1, 4) , and the enzyme, found in kidney, intestine, and bone, is induced by 1,25-(OH)(2)D(3) itself, thus programming the hormone's own breakdown(5, 6, 7) .

1,25-(OH)(2)D(3) activates transcription of the 24-hydroxylase gene by binding to its receptor, which in turn binds to specific response elements (DRE) that have been located in the 5`-upstream promoter region of the gene(8, 9, 10) . The consensus for DREs consists of 2 half-sites of 6 bp each separated by 3 nonspecific base pairs(2) . Most genes regulated by 1,25-(OH)(2)D(3) so far identified show considerable basal activity in the absence of 1,25-(OH)(2)D(3)(11, 12, 13) , while the highly regulated 24-hydroxylase is virtually without basal activity in the absence of 1,25-(OH)(2)D(3)(10, 14) . Two DREs have recently been identified within the first 300 bp 5` from the transcription start site(14, 15) . For all other genes regulated by 1,25-(OH)(2)D(3) so far elucidated, only one DRE located about 500 bp 5` to the start site has been found(11, 12, 13, 16, 17, 18) .

The present study demonstrates the synergistic importance of the DREs in this promoter and the requirement of a fifth half-site 3` from DRE closest to the transcriptional start site. Furthermore, we determined the relative affinities of both DREs for the VDR.


MATERIALS AND METHODS

Synthetic Oligonucleotides

Several synthetic oligonucleotides were generated by a DNA synthesizer (ABI, La Jolla, CA) as shown in Fig. 1A. These oligonucleotides were designed to have XbaI termini for labeling and subcloning. The larger oligonucleotides (over 100 bp) were amplified by polymerase chain reaction using the primers shown in Fig. 1B. The 24-hydroxylase promoter region was used as template (14) . DRE(1) and DRE(2) separated by 93 bp of nonspecific DNA were constructed using primers shown in Fig. 1C and chicken 24-hydroxylase cDNA^2 as template. The DRE sequences were included in the primers as shown in Fig. 1.


Figure 1: Synthetic Oligonucleotides.



Recombinant Reporter Constructs and Cell Transfection

The recombinant reporter gene constructs were obtained by cloning the oligonucleotides mentioned above into the XbaI site of the pBLCAT2 vector(19) . 2.5 µg/60-mm dish of these constructs were transfected into NRK-52E rat kidney cells using the Lipofectin method (Life Technologies, Inc.); the Lipofectin:DNA ratio was 3:1. Transfected cells were dosed with 1,25-(OH)(2)D(3) to a final concentration of 10 nM and incubated for 20-24 h. The cells were then harvested, and the activity of the chloramphenicol acetyltransferase (CAT) enzyme was measured as described before(14) .

Determination of Dissociation Constant (K)

All K(d)determinations were done by gel retardation assays. Probes were prepared synthetically or amplified by polymerase chain reaction as explained above. Labeling of each probe was done by repairing XbaI-recessed ends utilizing [alpha-P]dCTP (DuPont NEN) and the Klenow fragment of DNA polymerase (Promega). The probe obtained by polymerase chain reaction using chicken 24-hydroxylase cDNA was end-labeled utilizing [-P]dATP (DuPont NEN) and the T4 polynucleotide kinase (Promega). The labeled probes were purified by polyacrylamide gel electrophoresis followed by electroelution. The final probe concentrations after purification were 0.5 or 1 ng/µl. Binding of the VDR to the labeled DNA probes was performed as previously described (11) . Briefly, porcine intestinal nuclear extract containing VDR and the labeled DNA were incubated at 4 °C for 2 h, the time needed for maximal binding. The final KCl concentration was 75 mM, the concentration at which maximal binding occurs in the absence of 1,25-(OH)(2)D(3). When 1,25-(OH)(2)D(3) was added to the reaction, determinations were made at both 75 and 150 mM KCl, the latter being the concentration at which maximal binding occurs in the presence of 1,25-(OH)(2)D(3). The VDRbulletDNA complexes and unbound DNA were quantitated from the dried gels using the Betascope Analyzer (Betagen, Waltham, MA). All affinity measurements were calculated using computer-assisted graphics.


RESULTS

Fig. 2shows the structure of each pBLCAT2 construct used and their respective reporter gene activity induced by 1,25-(OH)(2)D(3). The entire DRE unit including 5`-half-sites produced a dramatic stimulation of CAT activity. Since this includes the DRE at -262 and the 3 half-sites at -154, the contribution of each was examined. The half-site at -262 is responsive to 1,25-(OH)(2)D(3) by itself but to a much lesser degree than the entire DRE system. The DRE at -154 is not responsive to 1,25-(OH)(2)D(3) by itself but requires the presence of a third half-site located at the 3`-end. All 3 half-sites are required for 1,25-(OH)(2)D(3)-dependent activity as shown in Fig. 3.


Figure 2: Ratio of CAT activities of transfected cells dosed with 1,25-(OH)(2)D(3) (+D) over transfected cells dosed with vehicle (-D). Each construct contains the thymidine kinase (tk) promoter and one or two DREs, drawn as arrows and labeled with letters representing each half-site. The direction of the arrows indicates that the consensus sequence resides on the antisense strand. DRE(1) (a . . . b) is located between -262 and -238, and DRE(2) (e . . . c . . . d) is located between -154 and -125 upstream of the transcription start site. The sequence of each DRE is indicated. The 93 bp that separate the two DREs represent the separation in the natural promoter. For comparative reasons, the activity of the construct containing its own promoter and 1400 bp upstream was also included (last line).




Figure 3: CAT activities resulting from different combinations of half-sites of DRE(2) (-154 to -125). The smallletters correspond to the half-sites depicted in Fig. 2. Activities are expressed as total counts of acetylated chloramphenicol obtained in 19 h on the Betascope analyzer. All reactions contained 100 µg of protein. The CAT assays were carried out as explained under ``Materials and Methods.''



In the intact promoter, a 93-base pair segment separates the two DREs. When this is deleted, the transactivation by 1,25-(OH)(2)D(3) is unchanged. Our results suggest that these two DREs containing a total of 5 half-sites account for most if not all of the transactivation potential of the intact promoter of the rat 24-hydroxylase. The somewhat higher +D/-D ratio seen with the intact 24-hydroxylase promoter and a promoterless CAT reporter is likely because of the lower base-line activity found in the cells without 1,25-(OH)(2)D(3).

K(d) values were determined for each DRE separately and together with their natural flanking sequences (separated by 93 bp) (Table 1). Multiple determinations were done, and a representative gel shift for each fragment is shown in Fig. 4. Increasing amounts of the indicated DNA probes were incubated with 24 fmol of VDR. Fig. 4shows the only complexes detected in the assays. The amount of complex formed was plotted versus the amount of unbound probe as shown in Fig. 5, A-C. These saturation plots show that the DREs separately (Fig. 5, A and B) reach saturation at a 250-fold higher concentration than both together (Fig. 5C ). The dissociation constants (K(d)) were calculated from Scatchard analysis as shown in the accompanying plots in Fig. 5(D-F)(20) . All Scatchard plots in Fig. 5show biphasic binding. Two K(d) values were calculated for each fragment. Table 1presents the K(d) determined for the DREs by themselves, their half-sites, and the entire D-responsive region of the 24-hydroxylase promoter. No cooperative binding of the VDR to the 2 half-sites of the DREs was detected (Fig. 6A), which could have explained the biphasic binding; there also seemed to be no cooperativity of VDR binding between both DREs (Fig. 6B). The tightest binding was observed with the intact DRE system. Substitution of the 93-bp fragment separating the two DREs with an irrelevant chicken DNA had little effect. The DRE at -268 to -238 had approximately [1/100] the binding affinity of the two DRE systems, while the DRE at -154 to -125 had [1/500] the affinity. Half-sites were bound poorly or about 10,000 times lower than the DRE complex and were not responsible for the biphasic binding pattern, since they seemed to have equal binding affinities for the VDR.




Figure 4: Electrophoretic gels of DREs and porcine intestinal nuclear extracts. A, DRE(1) (-262 to -238); B, DRE(2) (-154 to -134); and C, -260 to -136. These were the only complexes detected in the assay. The total concentrations loaded per lane (free + bound) is indicated above each lane. Radiolabeled probe was the only source of DNA in lanes containing less than 10 nM. At higher concentrations, [1/10] the amount was radiolabeled, and the rest was nonradioactive, resulting in a 10times dilution of the labeled DNA by unlabeled DNA. Gel retardation assays were carried out as explained under ``Materials and Methods.''




Figure 5: Representative saturation curves (A-C) with respective Scatchard plots (D-F) are shown for A and D (DRE(1), -262 to -238), B and E (DRE(2), -154 to -134), and C and F (-260 to -136). Average K(-1/slope) for several determinations are shown in Table 1. Data points were obtained from gel retardation assays described under ``Materials and Methods.''




Figure 6: Binding properties of increasing amounts of receptor in the presence of fixed amounts of probe. A, DRE(1) and DRE(2) assayed separately; B, DRE(1) and DRE(2) separated by their natural promoter sequence.



The K(d) values in the presence of ligand were also determined, and these values are also shown in Table 1. Binding proved to be 2-3-fold weaker in the presence of 1,25-(OH)(2)D(3), regardless of the salt concentration at which they were determined. However, the salt concentration at which maximal binding was achieved was higher (75 mM KCl versus 150 mM KCl) (data not shown). The fact that, in the absence of ligand, less salt is needed to destabilize the DNA-receptor complex would suggest that this complex is less stable than the complex with 1,25-(OH)(2)D(3), yet the dissociation constant is 2-3 times lower, which indicates greater affinity. We have no explanation for this interesting anomaly.

We then compared K(d) values with their respective 1,25-(OH)(2)D(3)-dependent transcriptional activities ( Fig. 2and Table 1). There was no such correlation between transcriptional activity and K(d) for any of the DNA fragments.


DISCUSSION

This report demonstrates that the promoter region of the 1,25-(OH)(2)D-24-hydroxylase gene of the rat contains the most powerful vitamin D-responsive element system reported to date. This system includes two distantly separated response elements, one of which contains 2 half-sites separated by 3 nonspecific bases in accordance with Umesono et al.(21) , and the other response element contains 3 half-sites that are essential for the transactivation activity by 1,25-(OH)(2)D(3) and its receptor. The DRE site closest to the transcriptional start site contains 3 half-sites, each separated by 3 base pairs, which is an unusual arrangement for vitamin D-responsive genes.

The most significant fact, however, is that there are two distinct D-responsive elements found in the promoter region of this gene, and both are required for maximal transactivation activity. These two DREs are separated by a 93-base pair fragment, which can be deleted without significantly reducing the transactivation potential of the system.

It is important to note that the presence of two hormone-responsive elements in the same promoter is unique for 1,25-(OH)(2)D(3) but not unusual, since, for example, the vitellogenin gene has two estrogen response elements(22) , while the mouse mammary tumor virus long terminal repeat contains multiple copies of the glucocorticoid response elements(23) .

In dissecting the transactivation potential of this system, our results demonstrate that the DRE, more distal to the transcriptional start site, is capable of responding in the reporter gene system to 1,25-(OH)(2)D(3). The DRE proximal to the transcriptional start site did not respond to 1,25-(OH)(2)D(3) until a third half-site was included. Each of the DREs alone could not approach the activity of the entire system. Finally, it is apparent from our results that the total 5 half-sites can account for virtually all of the transactivation potential of the VDR system found in the intact 24-hydroxylase promoter. Since the intact promoter was placed in a promoterless reporter gene system, the results are not entirely comparable with the data obtained using a reporter gene system containing the thymidine kinase promoter used to analyze fragments and constructs. Furthermore, the 24-hydroxylase promoter in the promoterless CAT reporter system shows low basal activity, which accounts for the higher ratio of +D/-D response.

Because of these findings, we attempted to learn whether the transactivation of each of these elements is related to their ability to bind to the receptor and the accessory protein. The K(d) values determined for each component of the VDR system did not correlate with its activity in the reporter gene system. However, the tightest binding occurred with the intact double responsive element system, and it was this system that gave maximum transactivation. In general, when examining the many oligonucleotides studied, no correlation between binding and transactivation was detected.

The 24-hydroxylase VDR system found in the promoter region represents an important new tool in deciphering the molecular mechanism whereby 1,25-(OH)(2)D(3) elicits an increased transcription of a target gene. We have also been able to utilize the DREs in the 24-hydroxylase promoter as an affinity resin for the isolation of the VDR and its nuclear accessory factor. This VDR system, therefore, represents an important advance in the molecular biology of vitamin D action.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Program Project Grant DK-14881, a fund from the National Foundation for Cancer Research, and a fund from the Wisconsin Alumni Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, University of Wisconsin-Madison, 420 Henry Mall, Madison, WI 53706. Tel.: 608-262-1620; Fax: 608-262-3453.

(^1)
The abbreviations used are: 1,25-(OH)(2)D(3), 1,25-dihydroxyvitamin D(3); DRE, vitamin D response element; CAT, chloramphenicol acetyltransferase; VDR, vitamin D receptor; bp, base pair(s).

(^2)
R. Ismail and H. F. DeLuca, unpublished results.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.