(Received for publication, July 29, 1994; and in revised form, October 12, 1994)
From the
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
. This system represents the most powerful of the DREs
reported to date.
Vitamin D 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)
D
, (
)the active hormonal
form of the vitamin. 1,25-(OH)
D
is produced by
two successive hydroxylations of vitamin D. First, it is hydroxylated
in the liver at C-25 to produce 25-OH-D
, the major
circulating form of the vitamin. Subsequently, 25-OH-D
is
further hydroxylated in the kidney by the 1
-hydroxylase to produce
1,25-(OH)
D
(1, 3) . Both
25-OH-D
and 1,25-(OH)
D
can be
further hydroxylated by 24-hydroxylase to produce
24,25-(OH)
D
and
1,24,25-(OH)
D
, 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)
D
itself, thus
programming the hormone's own
breakdown(5, 6, 7) .
1,25-(OH)D
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)
D
so far identified show considerable basal activity in the absence
of
1,25-(OH)
D
(11, 12, 13) ,
while the highly regulated 24-hydroxylase is virtually without basal
activity in the absence of
1,25-(OH)
D
(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)
D
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.
Figure 1: Synthetic Oligonucleotides.
Fig. 2shows the structure of each pBLCAT2 construct
used and their respective reporter gene activity induced by
1,25-(OH)D
. 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)
D
by itself but to a
much lesser degree than the entire DRE system. The DRE at -154 is
not responsive to 1,25-(OH)
D
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)
D
-dependent activity as shown in Fig. 3.
Figure 2:
Ratio of CAT activities of transfected
cells dosed with 1,25-(OH)D
(+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
(a . . . b) is located
between -262 and -238, and DRE
(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 (-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)D
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)
D
.
K 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
) 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
values
were calculated for each fragment. Table 1presents the K
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 (-262 to
-238); B, DRE
(-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 10
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, -262 to -238), B and E (DRE
, -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 and DRE
assayed separately; B,
DRE
and DRE
separated by their natural promoter
sequence.
The K 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)
D
, 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)
D
,
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 values with their respective
1,25-(OH)
D
-dependent transcriptional activities
( Fig. 2and Table 1). There was no such correlation
between transcriptional activity and K
for any of
the DNA fragments.
This report demonstrates that the promoter region of the
1,25-(OH)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)
D
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)D
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)D
. The DRE proximal to the
transcriptional start site did not respond to
1,25-(OH)
D
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 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)D
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.