Structural Organization of the Human Vitamin D Receptor Chromosomal Gene and Its Promoter
Ken-ichi Miyamoto1,
Robert A. Kesterson1,2,
Hironori Yamamoto,
Yutaka Taketani,
Eri Nishiwaki,
Sawako Tatsumi,
Yoshio Inoue,
Kyoko Morita,
Eiji Takeda and
J. Wesley Pike
Department of Clinical Nutrition (K.-i.M., H.Y., Y.T., E.N.,
S.T., Y.I., K.M., E.T.) Tokushima University Tokushima 770,
Japan
Department of Molecular and Cellular Physiology
(R.A.K., J.W.P.) University of Cincinnati Cincinnati, Ohio
45267
 |
ABSTRACT
|
---|
The vitamin D receptor (VDR) is known to mediate
the pleiotropic biological actions of 1,25-dihydroxyvitamin
D3 through its ability to modulate the
expression of target genes. The regulation of this ligand-activated
cellular transcription factor is reported to occur at both
transcriptional and posttranslational levels. To begin to address the
molecular basis by which the VDR gene is regulated transcriptionally,
we report here an initial characterization of the human VDR gene and
its promoter. We isolated several overlapping
-phage and cosmid
clones that cover more than 100 kb of human DNA and contained the
entire VDR gene. The gene is comprised of 11 exons that, together with
intervening introns, span approximately 75 kb. The noncoding 5'-end of
the gene includes exons 1A, 1B, and 1C. Eight additional exons (exons
29) encode the structural portion of the VDR gene product. While
primer extension and S1 nuclease-mapping studies reveal several common
transcriptional start sites, three unique mRNA species are produced as
a result of the differential splicing of exons 1B and 1C. The DNA
sequence lying upstream of exon 1A is GC rich and does not contain an
apparent TATA box. Several potential binding sites for the
transcription factor SP1 and other activators are evident. Fusion of
DNA fragments containing putative promoter sequences upstream of the
luciferase structural gene followed by transient transfection of these
plasmids into several mammalian cell lines resulted in significant
reporter activity. Due to the size and complexity of the 5'-end of the
VDR gene, we examined the activity of a DNA fragment surrounding exon
1C. An intron fragment 3' of exon 1C conferred retinoic acid
responsivity when fused to a reporter gene plasmid, suggesting a
molecular mechanism for the previously observed ability of retinoic
acid to induce the VDR. The recovery of the gene for the human VDR will
enable further studies on the transcriptional regulation of this gene.
 |
INTRODUCTION
|
---|
The biological actions of 1,25-dihydroxyvitamin D3
[1,25-(OH)2D3] that regulate transcriptional
events within the nucleus of target cells are mediated by the vitamin D
receptor (VDR)(1, 2, 3). This ligand-activated transcription factor
belongs to a superfamily of genes that encode receptors for the
steroid, thyroid, and vitamin A (retinoic acid and 9 cis-
retinoic acid) hormones as well as orphan receptor proteins for which
ligands have not yet been identified (4, 5, 6). Most members of this
receptor gene family have undergone considerable scrutiny during the
past several years, and significant progress has been made in
understanding their structure and function. Although genes that encode
proteins involved in mineral metabolism are a principal focus of VDR
action and its 1,25-(OH)2D3 ligand in
vivo (7, 8), the receptor is also capable of influencing genes
whose products regulate more fundamental processes of cellular
proliferation and differentiation (9, 10). The extensive biological
effects now ascribed to 1,25-(OH)2D3 can be
attributed, in large part, to the nearly ubiquitous tissue distribution
of the VDR as well as to the highly selective regulation of gene
expression by this hormone in specific cell types (1).
Many of the chromosomal genes for the nuclear receptor family members
have been cloned and their structural organization determined (11, 12, 13, 14, 15).
These genes are often more than 50 kb in length and are comprised of
multiple protein domain-associated exons separated by exceedingly large
introns. An exception to this appears to be the orphan receptors,
chicken ovalbumin upstream promoter-transcription factors
and ß,
which are encoded by genes that span 46 kb of DNA, contain only two
introns, and thus may represent ancestral members of this superfamily
of genes (16). The promoter regions of several of the receptor genes
have been characterized. They often appear to resemble housekeeping
genes, and many are embedded in GC-rich islands (11, 13). The absence
of a TATA box is a common feature of several of these genes as is the
existence of multiple start sites for transcription. In some cases,
mRNA transcripts are produced from more than one promoter (17, 18); in
others, transcripts are differentially spliced to create unique mRNA
species that encode functionally distinct receptor isoforms (19, 20).
Thus, nuclear receptor genes can be typified by the general structural
organization of the encoding exons as well as through the overall
characteristics of many of their promoters.
Transcriptional regulation of nuclear receptor gene expression is
clearly evident. While receptors for metabolites of vitamin D and
vitamin A, as well as the glucocorticoids are widely expressed (21),
receptors for estrogen, progesterone, and androgens display a more
restricted tissue-specific expression profile (22). Determinants of
restricted expression of these genes in tissues and cells as well as
the mechanisms that lead to activation of otherwise silent receptor
genes during development or during the differentiation of specific cell
types are generally not known. Expressed genes are, however, regulated
by a number of nonpeptides hormones as well as peptide hormones, growth
factors, and cytokines. Transcriptional autoregulation of receptor
genes by their respective gene products through cis elements
localized adjacent to cognate promoters is not uncommon. The
ß-retinoic acid receptor gene (RARß), for example, is autoregulated
through a ß-retinoic acid response element located immediately
adjacent to its promoter (23, 24) and accounts for substantial
up-regulation of RARß by retinoic acid both in vivo and
in vitro (25, 26).
The VDR is regulated at both transcriptional and posttranslational
levels. VDR gene expression is up-regulated in NIH-3T3 mouse
fibroblasts through activation of the protein kinase A pathway (27) and
down-regulated through activation of the protein kinase C pathway (28).
Both of these signal transduction pathways mediate the actions of PTH
on target cells (29) and thus may represent homeostatic mechanisms that
ultimately control cellular sensitivity to
1,25-(OH)2D3. Other growth factors and
cytokines are also known to regulate VDR gene expression, possibly
through the above pathways. Estrogens (30), thyroid hormone (31),
glucocorticoids (32), and retinoic acid (33, 34) are likewise able to
alter VDR mRNA levels in what appear to be tissue-specific patterns of
expression. Interestingly, both cell cycle (35) and the differentiation
state of cells in culture (36) influence the extent of VDR mRNA
expression. The level of the VDR under these circumstances may play a
regulatory role in the control of cellular proliferation and
differentiation by the vitamin D hormone in a wide variety of cell
types. Finally, homologous up-regulation of VDR mRNA by
1,25-(OH)2D3 has also been demonstrated both
in vitro and in vivo, again in a very
tissue-specific fashion (37, 38, 39). Whether this regulatory action occurs
directly as a result of VDR interaction with a
cis-element(s) at the 5'-end of the VDR gene, analogous to
the interaction of RARs with the RARß gene promoter, or indirectly as
a result of the induction or activation of other transcription factors
remains to be demonstrated.
To gain a better understanding of the molecular mechanisms by
which the VDR is regulated at the transcriptional level, we cloned the
chromosomal gene for the human VDR. We report here on structural
characterization and preliminary functional activity of its promoter.
The single gene for human VDR spans more than 75 kb of genomic DNA and
contains 11 exons. Three exons make up the 5'-noncoding region, and the
eight additional exons encode the structural component of the VDR. The
promoter is characterized by the lack of a TATA box initiator, its
GC-rich nature, and the presence of putative binding sites for SP1 and
a variety of transcription factors. It directs the transcription of at
least three VDR mRNA transcripts in kidney that appear to arise from
the differential splicing of 5'-noncoding exons. The promoter also
directs the transcription of a chimeric gene when fused upstream of an
expression plasmid containing the luciferase structural gene. The
recovery of the human VDR gene and these initial studies provide the
basis for further detailed examination of the transcriptional
regulation of the VDR gene. This effort is particularly warranted in
view of the central role of the VDR in
1,25-(OH)2D3 action and the potential
therapeutic role of 1,25-(OH)2D3 and
1,25-(OH)2D3 analogs in blocking cellular
proliferation and promoting the differentiation of tumor cells (40, 41).
 |
RESULTS
|
---|
The Structure and Sequence of hVDR mRNAs
We established first the authenticity of the 5'-end of the hVDR
mRNA sequence reported initially by Baker et al. (42) using
the 5'-RACE (rapid amplification of cDNA ends) PCR technique. Human
kidney RNA was employed as template to prepare first-strand cDNA, and
the latter was amplified using an anchored oligonucleotide (primer A)
as well as a downstream primer (primer B2) corresponding to hVDR cDNA
sequence as outlined in Fig. 1A
. Primary amplification
was followed by a secondary amplification as described in
Materials and Methods using primers A and B1. The cloning
and DNA sequence analysis of the resulting products revealed two
individual transcripts. As observed in Table 1
, the
sequence of the first (type 1) was identical to that reported by Baker
et al. (42) with the exception that the sequence of the DNA
product extended some 45 bp upstream. This suggests that the original
cDNA sequence reported by Baker et al. (42) was incomplete
at the 5'-end by 45 bases and that the type 1 PCR product might
represent the authentic 5'-end of the hVDR mRNA. The second transcript
(type 2) was identical to the type 1 transcript (see Table 1
) with the
exception that a 121-bp insert was discovered lying between nucleotides
-84 and -83 relative to the translation start site reported by Baker
and colleagues (42). An additional amplification of kidney RNA was then
carried out using primer A1 complementary to the authentic 5'-end of
the mRNA established above and primer B2 as illustrated in Fig. 1A
.
Cloning and sequence analysis of the products of this reaction revealed
the previous two mRNAs as well as a third (type 3). As documented in
Table 1
, type 3 was similar to type 1 with the exception that nts -83
to -3 relative to the sequence of Baker et al. (42) were
deleted. The failure of primer B1 to hybridize to type 3 transcripts
clearly prevented detection of type 3 in the original primary
amplification described above. The organization of the three
transcripts are illustrated in Fig. 1B
relative to the sequence of
Baker et al. (42). The nature of these transcripts, one of
which contains an insertion and the other a deletion, suggests that the
5'-end of the hVDR gene contains four exons, three of which are
exclusively noncoding exons, and that at least two of these exons may
undergo unique splicing events in the kidney. Based upon these data, a
fourth unique transcript containing a deletion of exon 1C is
possible.

View larger version (21K):
[in this window]
[in a new window]
|
Figure 1. Analysis of hVDR mRNA Transcripts Using 5'-RACE
A, Location of oligonucleotide primers used for DNA amplification. The
shaded rectangle illustrates portions of the hVDR
transcript originally reported by Baker et al. (42 ),
whereas the single line indicates additional portions of
the hVDR mRNA transcript revealed through 5'-RACE. The numbering
above the diagram corresponds to that of Baker et
al. (42 ) whereas the numbering below corresponds
to the largest hVDR mRNA transcript that was identified. The location
of DNA primers used to characterize the hVDR mRNA transcripts by
5'-RACE are indicated relative to the largest unspliced hVDR mRNA
transcript identified. The anchor primer (corresponding to vector
sequence) was obtained from Life Sciences. B, The three hVDR mRNA
transcripts identified through the 5'-RACE technique are illustrated
(type I, II, and III) relative to the transcript identified by Baker
et al. (42 ). Type I and type III transcripts contain
deletions that correspond to proposed exon 1B (type I) and exons 1B and
1C (type III), respectively. The type III transcript contains all the
proposed exons: 1A, 1B, and 1C. Exon 2 (43 ) is common to all
transcripts. The type IV transcript is hypothetical. The illustration
is numbered relative to the full-length transcript (type II).
|
|
-Phage Genomic Clones
Having characterized the 5'-end of the hVDR mRNA, we used
the 5'-RACE-derived DNA fragments to screen a
-phage genomic library
and recovered several positive clones. Three clones were mapped using
the restriction enzymes BamHI, EcoRI, and
SacI, and relevant probe-reactive fragments were sequenced.
As observed in Fig. 2
,
-clones 1 and 2 represent
overlapping clones that contain exons 1A and 1B, which are in turn
separated by an intron of approximately 5 kb.
-Clone 3 did not
overlap
-clone 2 but contained exon 1C and exon 2. Exon 2 contained
the start site of translation beginning 3 bp downstream of the 5'-end
of the exon. The intron located between exons 1C and 2 was determined
to be approximately 4 kb, but the lack of contiguity between
-clones
2 and 3 prevented determination of the size of the intron located
between 1B and 1C. The presence of these four exons is entirely
consistent with the data obtained from analysis of the hVDR mRNA. We
designated the first three exons 1A, 1B, and 1C to be consistent with a
preliminary description of the organization of this gene (43).
Cosmid Genomic Clones
In view of the lack of contiguity between
-clones 2 and
3, and to recover the remainder of the hVDR gene, we screened a human
liver genomic library prepared in the cosmid vector pCV109 (44)
sequentially with several nick-translated hVDR cDNA probes. Screening
resulted in the recovery of four unique individual human DNA cosmid
clones designated phVDRG1, 3, 4, and 11. As illustrated in Fig. 3A
, EcoRI, KpnI, XhoI,
and SalI restriction endonucleases were used initially to
map each of the respective clones and revealed that they represent
overlapping fragments of 40 to 50 kb which together span approximately
93 kb of contiguous genomic DNA.

View larger version (26K):
[in this window]
[in a new window]
|
Figure 3. Organizational Map of the Complete hVDR Chromosomal
Gene
A, Organizational map of the complete hVDR chromosomal gene. A human
liver cosmid library was screened with hVDR cDNA probes to obtain four
clones (phVDRG1, phVDRG4, phVDRG3, and phVDRG11) that spanned the
entire hVDR gene. Restriction mapping and Southern blot analysis
revealed the overlapping nature of the clones as well as the relative
position of each exon within the clone as indicated. Relevant
restriction fragments (A5.0, B4.2, C2.2, D4.4, E8.0, F5.5, G5.6, H2.7,
and I7.0) indicated as rectangles were subcloned and
subjected to high resolution restriction mapping and sequencing. X,
XhoI; K, KpnI; E, EcoRI.
The positions of the hVDR exons, which are numbered E1A, E1B, E1C,
E2-E9, are indicated by the black bars. B, Southern blot
analysis of phVDRG3 and phVDRG11 DNA and human genomic DNA. Overlapping
phVDRG clones 3 and 11 were subjected to restriction digestion with
EcoRI, KpnI, or both enzymes as indicated
(left). Human genomic DNA was subjected to simultaneous digestion with KpnI and EcoRI
(right). Southern blot analysis was carried out as
described in Materials and Methods and hybridized to the
hVDR3 probe. Bands D-H identified both in genomic DNA and within the
clones represent the DNA fragments schematically illustrated in A. Band
C was weakly evident on the autoradiogram but not reproducible in the
figure. C, Structural organization of the human chromosomal vitamin D
receptor gene. The structural organization of the human VDR gene locus
(DNA) comprising 11 exons (1A, 1B, 1C, 2 through 9) spanning
approximately 75 kb of DNA is depicted. A 10 kb scale is indicated to
the right. The location of exons relative to the mRNA
transcript of 4800 nucleotides (mRNA) and the encoded VDR protein of
427 amino acids (hVDR) is illustrated. With regard to the hVDR mRNA,
negative numbers indicate 5'-noncoding nucleotides, and positive
numbers indicate protein encoding nucleotides beginning with +1
indicated by Baker et al. (42 ) as well as
3'-untranslated sequences. Numbers below the hVDR
protein indicate the amino acid residue boundaries of shadedhomology domains. Regions of functionality are designated A/B,
C, D, and E/F as described in Ref. 76.
|
|
Three unique hVDR cDNA probes (see Materials and Methods)
were used to determine the extreme 5'- and 3'-boundaries of the VDR
gene within this locus and to determine the relative orientation of
individual DNA restriction fragments located within these four clones
with respect to the hVDR mRNA. Southern blot hybridization analysis
with these respective VDR cDNA probes revealed that clones G1, G3, and
G11 likely contained exons comprising the entire VDR chromosomal gene.
Subsequent detailed hybridization analysis of G3 and G11 DNA digested
with EcoRI, KpnI, or EcoRI and
KpnI utilizing hVDR cDNA probe 3 (nt +10 to
+2012 which includes the entire normal hVDR open reading
frame) revealed the presence of eight hybridizable DNA fragments
spanning a total of 55 kb (Fig. 3B
, left). The locations of
these fragments (identified as B4.2, C2.2, D4.4, E8.0, F5.5, G5.6,
H2.7, and I7.0) in relationship to the genomic cosmid clones are
depicted in Fig. 3A
. Importantly, each of these DNA restriction
fragments was unequivocally identified in human genomic DNA after
high-stringency Southern blot hybridization analysis with a combination
of the cDNA probes indicated above. Six of these fragments (C-H) are
detected after analysis of EcoRI- and
KpnI-digested human DNA with probe 3 (Fig. 3B
, right).
The position of exon 1A of 77 bases in genomic clone phVDRG1 was
verified through hybridization screening with an oligonucleotide
corresponding to a portion of exon 1A. DNA fragment A was sequenced to
identify the first exon located as illustrated in Fig. 3A
. Exons 1B
(121 bases), 1C (81 bases), and 2 (148 bases) located in this clone
were mapped relative to exon 1A, further defining the approximately 20
kb size of the intron located between exons 1B and 1C (see figure). DNA
fragments B and C (see Fig. 3A
) were cloned and sequenced to identify
the intron/exon boundaries of exons 1C and 2. Exon 1B was positioned
relative to exon 1A in
-clone 1.
Additional DNA fragments identified through hybridization analysis were
isolated, subcloned, and either partially or completely sequenced to
determine the precise location of downstream VDR exons. As observed in
Fig. 3C
, the gene is split into seven additional exons (exons 3 through
9) of 131, 185, 121, 172, 152, 117, and 3466 bp, which, together with
the upstream exons, form the authentic full-length hVDR gene. The gene
itself, however, spans some 75 kb of DNA, the majority consisting of
introns whose sizes, boundaries, and locations within the mature hVDR
mRNA are documented in Table 2
. A comparison of the
sequences of the intron/exon boundaries indicates that each conforms to
a canonical splice consensus sequence typical of most eukaryotic
genes.
The relative organization of the exons and their locations with
respect to domains located in the hVDR protein (45) is depicted in Fig. 3C
. Exon 2 contains 2 bp of noncoding sequence, the translation
initiation codon, and the N terminal of the two dissimilar DNA-binding
Zn++ finger modules. Exon 3 lies downstream of a 15-kb
intron and encodes the second DNA-binding zinc module. Exons 46 are
clustered together; exons 4 and 5 encode a region that serves a
hypothetical hinge function between DNA- and steroid-binding portions
of the receptor protein, while exon 6 encodes the remainder of the
hinge and the first portion of the steroid-binding domain. Finally,
clustered exons 79 encode the C-terminal half of the VDR, with exon 9
containing the final 85 amino acids as well as the remaining
approximately 3200 bp that constitute the large 3'-noncoding
sequence.
An Exon 2 Translation Site Polymorphism
Exon sequences within the VDR gene derived here were compared with
those of the human VDR cDNA recovered from the T47D breast cancer cell
line and reported by Baker et al. (42). Exon sequences
corresponded to those found in the hVDR cDNA with one exception, a T to
C transition that eliminated the most 5'-ATG codon within the T47D VDR
mRNA (see Table 2
). The likely result of this mutation, which creates a
potential polymorphic FokI site within exon 2, is the
utilization of a second in-frame translation codon beginning 10
nucleotides downstream that ultimately encodes a potentially
foreshortened receptor protein of 424 amino acids. Evaluation of
additional human DNA samples via DNA amplification techniques revealed
that this feature of the cloned gene was not unique to the genomic
library from which the hVDR gene was recovered (data not shown). More
importantly, perhaps, the existence of an hVDR mRNA of the latter type
has been demonstrated recently by Saijo et al. (46). Despite
these observations, however, it will be necessary to confirm that two
proteins that differ in molecular mass by only three amino acids are in
fact translated in human tissues or cells.
The hVDR Promoter
We sequenced a DNA fragment extending 5' of the start site
of transcription containing the putative promoter for hVDR (Fig. 4
). While this promoter lacks
consensus TATA or CAAT boxes, the region is GC rich
with five binding motifs for the transcription factor SP1 lying between
nucleotide (nt) -72 and -34 relative to the transcription start site
(47). Potential binding sites for other transcription factors are also
evident (48). In addition, five AGGTCA-like sequences, which represent
potential nuclear receptor-binding element half-sites (49), are located
between nt -1394 and -949. The functional relevance of these, as well
as of additional interesting sequences, will need to be determined.

View larger version (61K):
[in this window]
[in a new window]
|
Figure 4. Sequence Determination of the hVDR Gene Promoter
The nucleotide sequence of the hVDR gene promoter is documented. The
transcription start site is indicated as +1 beginning within the
boxed sequence. The locations of potential binding sites
for specific transcriptional regulators are underlined
and indicated. Transcription factors indicated include SP-1, AP-1,
AP-2, NFkB, GATA-1, Pit-1, and C/EBP. Potential binding sites for
nuclear receptors are also present.
|
|
Activity of the 5'-Flanking Region of the hVDR
Promoter
To evaluate the transcriptional capacity of the
hVDR promoter, we cloned a series of 5'-deletion fragments of the gene
into a luciferase reporter gene and transfected them individually into
two mammalian cell lines. We tested constructs beginning at -1.935,
-1.479, -1.221, -0.586, -0.464, -0.103, -0.034 kbp relative to
the start site of transcription. The downstream boundary of each
construct was located at the 3'-boundary of exon 1A at +71 relative to
the start site of transcription [-89 relative to the start site of
translation reported by Baker et al. (42)]. As observed in
Fig. 5A
, pVDE11.93 was capable of directing
significant luciferase expression when introduced by transient
transfection into HeLa cells. Each deletion construct likewise
exhibited substantial transcriptional activity, although this activity
varied and did not exhibit an obvious pattern. Constructs containing
proximal elements of the promoter (-464 and -103 relative to the
start site) displayed the most activity, suggesting the possibility
that elements upstream of -0.464 kb may confer a negative regulatory
function. 5'-Deletion of the hVDR promoter from -103 bp to -34 bp
that removed four of the five GC boxes near the initiator resulted in a
10-fold drop in activity. The latter result substantiates the
hypothesis that these elements play an important role in the activity
of the hVDR promoter and provide the basis for future studies.
Preliminary studies in Hela cells suggest that the 5'-flanking region
of the gene is not responsive to 1,25-(OH) 2D3
(data not shown).

View larger version (24K):
[in this window]
[in a new window]
|
Figure 5. Functional Analysis of the Human VDR Gene Promoter
A, hVDR promoter constructs pVDE11.93, pVDE11.48, pVDE11.22,
pVDE10.589, pVDE10.46, pVDE10.10, pVDE10.03, and the
promoterless control plasmid (pGL-2) were individually cotransfected
together with the normalization vector pCMV-ß-galactosidase as
indicated in Materials and Methods. The activities of
these constructs in Hela cells were determined after 64 h and
normalized to the activity of ß-galactosidase. Luciferase activity is
expressed as light units per second per mg of protein. Data are
means ± SEM of four independent experiments. B, DNA
fragments surrounding exon 2 as indicated in the figure were fused to
the pBL CAT2 or pBLCAT3 chloramphenicol acetyltransferase expression
vectors and transfected into rat ROS 17/2.8 cells as described in
Materials and Methods. Transfected cells were treated
with either vehicle or retinoic acid (10-6 M)
for 48 h after which the cells were harvested and extracts (100
µg protein) assayed for CAT activity. Each assessment represents the
mean ± SEM of a triplicate determination. The data
are representative of three separate experiments and are expressed as
the activity of the construct in the presence of retinoic acid compared
with the activity of the construct in the ligands absence (fold
induction).
|
|
A Retinoid-Responsive Region Lies Downstream of Exon 2
In view of the complexity of the hVDR gene, we examined
several DNA fragments surrounding exon 1C for their capacity to direct
transcriptional activity or to be regulated by hormones such as
1,25-(OH)2D3 or retinoic acid. All clones
examined exhibited transcriptional activity when cloned into the
promoterless chloramphenicol acetyltransferase (CAT) vector pBLCAT3
(data not shown). As observed in Fig. 5B
, however, several constructs,
each containing intron sequence downstream of exon 2, were responsive
to treatment with retinoic acid (10-6
M). The constructs were uniformly unresponsive to
1,25-(OH)2D3 in this cell line (data not
shown). The ability of this fragment of DNA to transfer
retinoic acid response to the viral thymidine kinase promoter provides
further support for the possibility that the previously identified
regulation of VDR expression by retinoic acid (33, 34) is direct and
mediated via a cis-element located a significant distance
downstream of the authentic promoter. Further work will be required to
define the exact location of this cis-element.
 |
DISCUSSION
|
---|
We report here the structural organization of the human VDR gene
derived from several
-clones and four overlapping cosmid clones that
span the entire gene locus. The human gene comprises 11 exons that
together with the associated introns cover approximately 75 kb of DNA.
Three exons (exons 1A, 1B, and 1C) make up the 5'-noncoding leader
sequence of the largest of the hVDR mRNA species (type 2). An
additional eight exons (exons 29) encode the structural portion of
the gene product. This numbering system allows retention of the
numbering system assigned in an earlier preliminary analysis of the
gene (43) as well as that found in a body of literature related to the
existence of hVDR gene polymorphisms that has evolved recently (see
below). The multiple exonic structure and general organization of the
hVDR gene are comparable to that of many of the other steroid receptor
genes that have been characterized including the thyroid receptor (18),
progesterone receptor (11), estrogen receptor (12), androgen receptor
(14), and glucocorticoid receptor (13, 17). The promoter for this gene
is TATA-less and GC rich.
The organization of the hVDR gene indicates that separate exons encode
each of the zinc finger modules and that the 3'-boundaries of each of
these exons appear to be generally conserved within the nuclear
receptor family of genes. One exception may be chicken ovalbumin
upstream promoter-transcription factor
and ß, for which both
finger modules appear to be encoded by a single exon (16). Although the
two zinc modules within these proteins appear highly related
structurally, they are not equivalent topologically (50). Furthermore,
the function of each module is substantially different; the first zinc
module determines the specificity of DNA binding whereas the second is
more intimately involved in the protein-protein contacts that stabilize
the association through dimerization (51). Although it is possible that
the two exons encoding these modules evolved from a common ancestral
gene through duplication and subsequently diverged under a different
set of selective pressures, it is also possible that they evolved
independently. Three exons encode the hVDR hinge, whereas only two
encode this flexible region within the sex steroid receptor genes. It
is noteworthy that the VDR appears to contain an extended hinge region
relative to many of the other members of this gene family (4). The
function, if any, of these additional hVDR residues that are encoded by
insertion of exon 5 is unknown, although this region appears to be the
least conserved among VDRs from different species. Finally, exon 2 of
the hVDR is restricted to 21 amino acids upstream of the first zinc
module, placing the hVDR DNA-binding domain near the amino-terminal end
of the protein. This is unlike several of the other family members
where one or more relatively large exons lie upstream, relegating the
DNA-binding domain to a more central location within the protein (4).
The absence of this extended region within the hVDR, whose presence in
the larger receptors is associated with an important activation
domain(s) (52, 53, 54), implies a more complete reliance of the hVDR on the
carboxy-terminal activation function (AF2) or on an, as yet,
undescribed activation region.
Amplification of the 5'-end of the hVDR mRNA revealed the existence of
three separate transcripts in human kidney RNA. Because each contains
the identical start site and first exons, the transcripts are likely
derived from alternative splicing of two of the 5'-noncoding exons, 1B
and 1C, one of which eliminates exon 1B and the other which eliminates
both exons 1B and 1C. It is hypothetically possible that a fourth
transcript that does not contain exon 1C also exists, perhaps in
tissues other than kidney. Alternative splicing of the RAR, retinoid X
receptor, thyroid hormone receptor, and other receptors is a common
feature of this gene family of proteins. Unlike that which was observed
here, some splicing events lead to the productions of different
proteins with unique functions (19, 20). The relative abundance of each
of the three hVDR transcripts within the kidney is unknown; indeed, the
similarity in their size prevented their earlier detection by Northern
blot analysis. The existence and relative abundance of these hVDR
transcripts in other tissues as well as a determination of their
possible individual functions remain for future studies.
The promoter region of the hVDR lies in a GC-rich island and does not
contain a TATA box. In that respect, the hVDR gene is like certain
other steroid receptor gene promoters. Our analysis of this promoter
indicates a substantial capacity to direct transcription of a chimeric
reporter function. The most proximal region (-103 to -34) imparts
more than 80% of the activity of the promoter in HeLa cells and
appears to contain strong positive elements. A possible negative
contribution is evident between -586 and -464. The hVDR promoter
contains an array of putative binding sites for transcription factors
that mediate the activities of multiple pathways that serve to
transduce a variety of extracellular signals. For example, it is known
that the VDR is regulated by both PKA and PKC pathways (27, 28, 29, 55, 56)
that are in turn known to converge on several specific transcription
factors. These and other studies support the possibility that binding
sites upstream of the hVDR promoter may play modulatory roles in its
regulation, although future functional studies will be necessary to
confirm this hypothesis. At the very least, the presence of five GC
boxes immediately upstream of the start site of transcription suggests
a fundamental role for the transcription factor SP1 in the activation
of this gene (47). SP1 is known to interact both with a number of
cellular and viral promoters as well as with other transcription
factors such as NFkB (57, 58). Interestingly, the hVDR gene is
induced with retinoic acid at a site that lies downstream of exon 1C.
While a potential binding site for one or more of the retinoid
receptors has not been definitively localized, it is likely that this
site mediates the recognized ability of RA to induce the transcription
of VDR (33, 34). The existence and, more importantly, the location of
this site, together with the size of the introns lying between exons
1A, 1B, and 1C, suggest that it may prove difficult technically to
identify additional regulatory sequences. The inability to detect
vitamin D inducibility of the VDR gene at the promoter level currently
suggests that the autoregulatory actions of vitamin D are indirect. For
example, a recent study suggests that
1,25-(OH)2D3 can stimulate the expression of
c-fos in osteoblastic cells that might in turn stimulate
hVDR gene expression (59). Based upon the noted complexity at the
5'-end of the VDR gene, however, it is possible that
1,25-(OH)2D3-responsive portions of the gene
have yet to be defined.
During the analysis of the hVDR gene, we identified the existence of a
polymorphism associated with exon 2. This polymorphism leads to
synthesis of two different hVDR mRNAs which we detected utilizing
single-strand conformational analysis (46). One corresponds to the mRNA
sequence of Baker et al. (42) wherein the start site of
translation is situated at codon 1 and the second is associated with a
T to C conversion in this codon that results in potential translation
beginning at an in-frame ATG located downstream at codon 4. Translation
of such an mRNA would result in the production of an abbreviated
protein of only 424 amino acids. Two studies have recently reported the
distribution of these alleles in the human population (60, 61); the ATG
to ACG allele, which can be detected by the presence of a
FokI site (60), represents the more common form.
Interestingly, the frequency of this allele correlates with an increase
in bone density in two different human female populations (60, 61).
This observation suggests that the VDR products of these two alleles
may exhibit unique activities. Validation of this hypothesis, however,
will require additional population studies as well as the demonstration
that the two VDR alleles are indeed expressed in human tissues and that
they exhibit quantitative and/or qualitative differences in their
activity on bone or other tissues. As a first step, Arai et
al. (61) have shown that both proteins can be produced through
recombinant means in transfected COS cells. More importantly, the
capacity of these two gene products to direct transcription of a
vitamin D-sensitive chimeric gene in cotransfected cells is
quantitatively different. The smaller protein, whose corresponding
allele appears to correlate with increased bone density, exhibited
greater transcriptional activity. Additional studies will be necessary
to confirm these functional studies as well as the correlation between
bone mineral density and VDR alleles. Interestingly, some plasticity is
also apparent in these two codons in other species; while the mouse VDR
mRNA contains both ATGs (62), the rat VDR mRNA contains only the most
5'-ATG (63). Perhaps unique activities are also associated with these
mRNA products.
Additional polymorphisms have been identified within the hVDR
gene in the intron between exons 7 and 8 (BsmI and
ApaI) and within the 3'-noncoding sequences lying in exon 9
(TaqI) (64) (see Table 3). The presence of these restriction
fragment length polymorphisms has been reported to be associated with
population bone mineral density (65) and more recently with prostate
cancer (66). In the former case, this finding has not been widely
reproduced, suggestive of relatively weak linkage, and remains highly
controversial (67). In contrast to the FokI polymorphism
located in exon 2, it is unclear how the BsmI and/or
TaqI polymorphisms located in noncoding portions of the hVDR
gene might influence hVDR function. It is possible that they are linked
in some way either to the potentially functional exon 2 start site
polymorphism, to as yet unidentified allelic differences located within
the hVDR gene promoter, or to unrelated genes that impact bone
mineralization directly. Both the validity of the proposed associations
between hVDR polymorphisms and disease and, if proven true, the
mechanism(s) by which they impart disease remain to be established.
In conclusion, we report on the structural organization of the hVDR
chromosomal gene. The availability of this gene locus as well as
identification and cloning of its promoter will enable future studies
aimed at identifying molecular determinants of the VDRs expression. A
wealth of studies that describe the regulation of expression of the VDR
provide the backdrop and rationale for these impending studies.
 |
MATERIALS AND METHODS
|
---|
Messenger RNA Analysis
The sequence of the 5'-end of the human VDR mRNA was determined
utilizing the 5'-RACE system obtained from Life Technologies, Inc,
(Gaithersburg, MD) (68). Total RNA was isolated from human kidney (69, 70) and used to synthesize first-strand cDNA utilizing random hexamers
and oligo d(T) primers and Superscript RT (Life Technologies, Inc). Two
sequential PCR amplification reactions were employed to identify the
5'-ends of the hVDR mRNAs and to generate DNA fragments useful for
initial sequencing. As illustrated in Fig. 1A
, the first was performed
using common anchored primer A
(5'-CTGGTTCGGCCCACCTCTGAAGGTTCCAGAATCGATAG-3') and primer B2
(5'-CTTCCGCTTCAGGATCATCTCCCGC-3'), which corresponds to nt +309 to +333
relative to the translation start site of the human VDR cDNA of Baker
et al. (42). The second was performed with a portion of the
reaction products from the first amplification and used primer A and
primer B1 (5'-GCAGGGGGCAGGTAAGTGG-3'), which corresponds to nt -29 to
-11 relative to the start site of translation reported by Baker
et al. (42). Sequence determination of the PCR products of
these sequential reactions established the 5'-end of the hVDR mRNA
transcript, enabling a subsequent amplification of kidney RNA using
primer A1, which corresponds to the first 22 bases of the authentic
5'-end of the transcript and primer B2 (see Fig. 1A
). All amplified DNA
products were subcloned into pBluescript II SK (Strategene, San Diego,
CA) and sequenced using the SequiTherm Long-Read Cycle Sequencing
Kit-LC (EpiCentre Technologies, Madison, WI). All DNA sequencing was
performed using the fluorescence-based LI-COR model 4000L
sequencer.
Recovery and Analysis of hVDR Genomic Clones
A human EMBL 3 genomic library was screened with
32P-labeled PCR probes obtained after 5'-RACE of human
kidney mRNA using high-stringency hybridization conditions. Nylon
membrane replicas of approximately 1.5 x 106 plaques
were incubated for 2 to 3 h in 50% formamide, 5x Denhardts
solution, 0.75 M NaCl, 0.05 M
NaH2PO4, pH 7.4, 3 mM EDTA, 0.1%
SDS, and 0.1 mg/ml salmon sperm DNA. Hybridization was performed
overnight at 42 C with 5 x 105 cpm/ml labeled probe.
Membranes were washed for 1 h at 30 C in 0.3 M NaCl,
0.03 M sodium citrate, pH 7.0, and 0.05% SDS and then
twice for 1 h at 68 C in 0.15 M sodium citrate, pH
7.0, and 0.1% SDS. After exposure of the membranes to Kodak X-Omat
film, positive plaques were isolated, rescreened twice, and purified,
and then the inserts were isolated and subcloned into pBlueScript II
SK. DNA inserts were verified by Southern blot analysis, restriction
enzyme-mapped by routine methods, and portions were then sequenced
using the strategy outlined below.
A human liver genomic library prepared in the cosmid vector
pCV109 (44) was screened via colony hybridization techniques (71) using
nick-translated hVDR cDNA probes. HVDR probe 1 extended from -115 to
+145 nt relative to the sequence of Baker et al. (42); hVDR
probe 2 contained nucleotides +10 through +576 relative to the sequence
of Baker et al. (42); and hVDR probe 3 contained nucleotides
+10 through +2012 nt relative to the sequence of Baker et
al. (42). The latter probe represents the entire open reading
frame of the normal hVDR cDNA together with 731 nucleotides of
noncoding 3'-sequence. Nylon filter-immobilized DNA was prehybridized
at 68 C overnight in 6x NaCl-sodium citrate (SSC) (1x = 0.15
M NaCl2, 15 mM sodium citrate, pH
7), 2 mM EDTA/0.5% nonfat dry milk (wt/vol), and then
hybridized with 32P-labeled probes (initially probes 2 and
3) under identical conditions for 16 h. Filters were washed for
2 h with several changes of 0.15 M NaCl2,
15 mM sodium citrate, pH 7, 0.5% SDS at 68 C and then
autoradiographed overnight. Positive phVDRG cosmid clones were isolated
through three to four rounds of additional screening.
Human genomic DNA was prepared as described (71). The latter as well as
cosmid phVDRG DNA were transferred to nylon membranes, prehybridized
for 6 h at 68 C in 6x SSC, and then hybridized with the
appropriate 32P-labeled hVDR cDNA probes overnight. Filters
were washed for 2 h as above, and autoradiographed for 4 h
(cloned DNA) or 48 h (human genomic DNA). phVDRG DNA was isolated
via alkaline lysis and polyethylene glycol precipitation techniques as
previously described (71). The isolated DNA was mapped with restriction
endonucleases by routine techniques. The relative orientation of
specific DNA fragments of related size within the clones was determined
by sequential probing with hVDR cDNA probes 1, 2, and 3. DNA fragments
that hybridized with the hVDR cDNA probes were isolated, subcloned into
the pGEM 3 cloning vector (Promega Biotech, Madison, WI), and then
subjected to standard sequencing methods using Sequenase (US
Biochemical Corp., Cleveland, OH). The sequencing strategy we employed
involved determining the ends of each DNA fragment utilizing the T7 and
SP6 sequencing primers followed by extension of this sequence with
synthetic oligonuclotides complementary to the newly identified
sequence. As the relative position of hVDR gene exons emerged within
the DNA clones, synthetic oligonucleotides corresponding to hVDR cDNA
sequence either 5' or 3' to the identified exon were used as sequencing
primers. Intron/exon boundaries were identified using pairs of primers
that generated sequence from both strands. Orientation of the exon(s)
within the DNA fragment was achieved by identifying sequence overlaps
within the clone or by mapping a unique restriction site identified
within the newly obtained sequence.
hVDR Gene Promoter Analysis
hVDR promoter constructs were prepared beginning with an
approximately 3.2 kb SacI-HindIII fragment of the
hVDR gene extending from exon 1 upstream approximately -3200 bp. This
fragment (pVDE13.2) as well as additional fragments were cloned into
the HindIII site of the luciferase expression vector pGL-2
basic (Promega). Plasmids pVDE12.6, pVDE11.93, pVDE11.48,
pVDE11.22, pVDE10.59, pVDE10.46, pVDE10.10, and pVDE10.03
were similarly constructed from promoter fragments that contained
common 3'-ends but extended 2.6, 1.93, 1.22, 0.586, 0.462, 0.10, or
0.03 kb upstream, respectively. DNA restriction fragments were also
isolated from hVDR gene DNA surrounding exon 1C. These fragments
include exon 1C and the length of intron sequence lying both upstream
and/or downstream of exon 1C as indicated in the figure. The activity
of these fragments of DNA was assessed in promoterless pBL-CAT3 and
thymidine kinase promoter-containing pBL-CAT2 chloramphenicol
acetyltransferase expression vectors (72). The orientation and cloning
boundaries of all constructs were verified through DNA sequence
analysis.
Transient Transfection Analysis of hVDR Promoter Sequences
Hela cells were cultured in MEM supplemented with 10% FBS, 2
mM L-glutamine, penicillin (100 U/ml),
streptomycin (0.1 mg/ml), and nonessential amino acids. Rat
osteosarcoma ROS 17/2.8 cells were grown in Hams F12 medium
supplemented with 10% FCS containing penicillin (100 U/ml),
streptomycin (0.1 mg/ml), and nonessential amino acids as previously
described. Cells were transfected with DNA 24 h after passage
using either polybrene (73) (Sigma Chemical Co., St. Louis, MO) (ROS
17/2.8) or lipofectAMINE (Life Technologies Inc.) (HeLa cells). hVDR
gene DNA reporter plasmids (5 or 10 µg) were cotransfected together
with 1 µg ß-galactosidase normalization vector (1 µg), and the
activities of the enzymes CAT, luciferase, and/or ß-galactosidase
were evaluated in cellular extracts prepared 64 h following
transfection. Luciferase or CAT activities were determined as
previously described (65, 74) and normalized to the activity of
ß-galactosidase (75). All plasmids used for cellular transfections
were purified on Qiagen ion exchange columns.
 |
ACKNOWLEDGMENTS
|
---|
The authors wish to thank Savio Woo and Anthony Dilela for
providing the human cosmid library and to acknowledge Clark Huckaby,
Bert OMalley, Shigeaki Kato, and Keiichi Ozono for helpful
discussions.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. J. Wesley Pike, Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio 45267 or Dr. K. Miyamoto, Department of Clinical Nutrition, Tokushima University, Tokushima 770, Japan.
This work was supported by Grants-in-Aid for Scientific Research from
the Ministry of Education and Science, the Setsuro Fujii Memorial, the
Osaka Foundation for Promotion of Fundamental Research, Uehara Memorial
Foundation, Salt Science Research Foundation, and the NIH.
1 The contributions of the first two authors of this manuscript should
be considered equivalent. 
2 Present address: The Vollum Institute for Advanced Biomedical
Sciences. Oregon Health Sciences University. 3181 Sam Jackson Park
Road, Portland, Oregon 97201. 
Received for publication February 12, 1997.
Revision received March 24, 1997.
Accepted for publication March 26, 1997.
 |
REFERENCES
|
---|
-
Pike JW 1991 Vitamin D3 receptors: structure
and function in transcription. Annu Rev Nutr 11:189216[CrossRef][Medline]
-
Haussler MR, Jurutka PW, Hsieh J-C, Thompson PD, Selznick SH,
Haussler CA, Whitfield GK 1995 New understanding of the molecular
mechanism of receptor-mediated genomic actions of the vitamin D
hormone. Bone 17:33S38S[CrossRef]
-
Christakos S, Raval-Pandya M, Wernyj RP, Yang W 1996 Genomic
mechanisms involved in the pleiotropic actions of 1,25-dihydroxyvitamin
D3. Biochem J 316:361371[Medline]
-
OMalley BW 1990 The steroid receptor superfamily: more
excitement predicted for the future. Mol Endocrinol 4:363344[Medline]
-
Beato M, Herrliche P, Schutz G 1995 Steroid hormone
receptors: many actors in search of a plot. Cell 83:851857[Medline]
-
Mangelsdorf DJ, Evans RM 1995 The RXR heterodimer and orphan
receptors. Cell 83:841850[Medline]
-
Stein GS, Lian JB, Stein JL, Van Wijnen AJ, Montecino M 1996 Transcriptional control of osteoblast growth and differentiation.
Physiol Rev 76:593629[Abstract/Free Full Text]
-
Kerner SA, Scott RA, Pike JW 1989 Sequence elements in the
human osteocalcin gene confer basal activation and inducible response
to hormonal vitamin D3. Proc Natl Acad Sci USA 86:44554459[Abstract]
-
Abe E, Miyaura C, Sakagami H, Takeda M, Konno K,
Yammaki T, Yoshiki S,and Suda T 1981 Differentiation of mouse myeloid
leukemia cells induced by 1,25-dihydroxyvitamin D3. Proc
Natl Acad Sci USA 78:49904994[Abstract]
-
Liu M, Lee M-H, Cohen M, Freedman LP 1996 Transriptional
activation of the p21 gene by vitamin D3 leads to the
differentiation of the myelomonocytic cell line U937. Genes Dev 10:142153[Abstract]
-
Huckaby CS, Conneely OM, Beattie WG, Dobson AD, Tsai MJ,
OMalley BW 1987 Structure of the chromosomal chicken progesterone
receptor gene. Proc Natl Acad Sci USA 84:83808384[Abstract]
-
Ponglikitmongol M, Green S, Chambon P 1988 The cloned human
estrogen receptor contains a mutation which alters its hormone binding
properties. EMBO J 7:33853388[Abstract]
-
Encio IJ, Detera-Wadleigh SD 1991 The genomic structure of the
human glucocorticoid receptor. J Biol Chem 266:71827188[Abstract/Free Full Text]
-
Marcelli M, Tilley WD, Wilson CM, Griffin JE, Wilson JD,
McPhaul MJ 1990 Definition of the human androgen receptor gene
structure permits the identification of mutations that cause androgen
resistance: premature termination of the receptor protein at amino acid
residue 588 causes complete androgen resistance. Mol Endocrinol 4:11051116[Abstract]
-
Lehman JM, Hoffman B, Pfahl M 1991 Genomic organization of the
retinoic acid receptor gamma gene. Nucleic Acids Res 19:573778[Abstract]
-
Ritchie HH, Wang LH, Tsai S, OMalley MJ 1990 COUP-TF
gene: a structure unique for steroid/thyroid receptor superfamily.
Nucleic Acids Res 18:68576862[Abstract]
-
Strahle U, Schmidt A, Kelsy G, Steward AF, Cole TJ,
Schmid W, Schultz G 1992 At least three promoters direct expression of
the mouse glucocorticoid receptor gene. Proc Natl Acad Sci USA 89:67316735[Abstract]
-
Zahraoui A, Cuny G 1987 Nucleotide sequence of the chicken
proto-oncogene c-erb A corresponding to domain 1 of v-erb A. Eur J
Biochem 166:6369[Abstract]
-
Misrahi M, Loosfelt H, Atger M, Meriel C, Zerah V, Dessen
P, Milgrom E 1988 Organization of the entire rabbit progesterone
receptor mRNA and of the promoter and 5' flanking region of the gene.
Nucleic Acids Res 16:54595472[Abstract]
-
Lazar MA 1993 Thyroid hormone receptors: multiple forms,
multiple possibilities. Endocr Rev 14:184193[Medline]
-
Kastner P, Mark M, Chambon P 1996 Nonsteroid nuclear
receptors: what are genetic studies telling us about their role in real
life. Cell 83:859869
-
Beato, M, Herrlich P, Schutz G 1996 Steroid hormone receptors:
many actors in search of a plot. Cell 86:851857
-
Umesono, K, Giguere V, Glass CK, Rosenfeld MG, Evans RM 1988 Retinoic acid and thyroid hormone induce gene expression through a
common responsive element. Nature 336:262265[CrossRef][Medline]
-
de The H, Del Mar Vivanco-Ruiz M, Tiollais P, Stunnenberg
H, DeJean A 1990 Identification of a retinoic acid responsive element
in the retinoic acid receptor ß gene. Nature 343:177180[CrossRef][Medline]
-
Davis KD, Berrodin TJ, Stelmach JE, Winkler JD, Lazar MA 1994 Endogenous retinoid X receptors can function as hormone receptors in
pituitary cells. Mol Cell Biol 14:71057110[Abstract]
-
Allegretto EA, Shevde N, Zou A, Howell SR, Boehm MF, Hollis
BW, Pike JW 1995 Retinoid X receptor acts as a hormone receptor
in vivo to induce a key metabolic enzyme for
1,25-dihydroxyvitamin D3. J Biol Chem 270:2390623909[Abstract/Free Full Text]
-
Krishnan AV, Feldman D 1991 Cyclic adenosine
3'.5'-monophosphate upregulates 1,25-dihydroxyvitamin D receptor gene
expression and enhances hormone action. Mol Endocrinol 6:198206[Abstract]
-
Krishnan AV, Feldman D 1991 Activation of protein kinase-C
inhibits vitamin D receptor gene expression. Mol Endocrinol 5:605612[Abstract]
-
Krishnan AV, Cramer SD, Bringhurst RF, Feldman D 1995 Regulation of 1,25-dihydroxyvitamin D3 receptors by
parathyroid hormone in osteoblastic cells: role of second messenger
pathways. Endocrinology 136:705712[Abstract]
-
Levy J, Zuili I, Yankowitz N, Shany S 1984 Induction of
cytosolic receptors for 1,25-dihydroxyvitamin D3 in the
immature rat uterus by oestradiol. J Endocrinol 100:265269[Abstract]
-
Mahonen A, Pirskanen A, Maenpaa PH 1991 Homologous and
heterologous regulation of 1,25-dihydroxyvitamin D3
receptor mRNA levels in human osteosarcoma cells. Biochim Biophys Acta 188:111118
-
Chen TL, Cone CM, Morey-Holton E, Feldman D 1983 1,25-Dihydroxyvitamin D3 receptors in cultured rat
osteoblasts-like cells. Glucocorticoid treatment increases receptor
content. J Biol Chem 258:43504355[Abstract/Free Full Text]
-
Petkovich PM, Heersche JNM, Tinker DO, Jones G 1984 Retinoic
acid stimulates 1,25-dihydroxyvitamin D3 binding in rat
osteosarcoma cells. J Biol Chem 259:82748280[Abstract/Free Full Text]
-
Chen TL, Feldman D 1985 Retinoic acid modulation of
1,25(OH)2D3 receptors and bioresponse in bone
cells. Biochem Biophys Res Commun 132:7480[Medline]
-
Krishnan AV, Feldman D 1991 Stimulation of
1,25-dihydroxyvitamin D3 receptor gene expression in
cultured cells by serum and growth factors. J Bone Miner Res 6:10991107[Medline]
-
Zhao X, Feldman D 1993 Regulation of vitamin D receptor
abundance and responsiveness during differentiation of HT-29 human
colon cancer cells. Endocrinology 132:18081814[Abstract]
-
Costa EM, Hirst MA, Feldman D 1985 Regulation of
1,25-dihydroxyvitamin D3 receptors by vitamin D analogs in
cultured mammalian cells. Endocrinology 117:22032210[Abstract]
-
Pan LC, Price PA 1987 Ligand-dependent regulation of the
1,25-dihydroxyvitamin D3 receptor in rat osteosarcoma
cells. J Biol Chem 262:46704675[Abstract/Free Full Text]
-
McDonnell DP, Mangelsdorf DJ, Pike JW, Haussler MR, OMalley
BW 1987 Molecular cloning of complementary DNA encoding the avian
receptor for vitamin D. Science 235:12141217[Medline]
-
Abe J, Morikawa M, Miyamoto K, Kaiho S, Fukushima M, Miyaura
C, Abe E, Suda T, Nishii Y 1987 Synthetic analogues of vitamin
D3 with an oxygen atom in the side chain skeleton. A trial
of the development of vitamin D compounds which exhibit potent
differentiation-inducing activity without inducing hypercalcemia. FEBS
Lett 226:5862[CrossRef][Medline]
-
Binderup L, Latini S, Binderup E, Bretting C, Calverly MJ,
Hlansen K 1991 20-Epi-vitamin D3 analogues: a novel class
of potent regulators of cell growth and differentiation. Biochem
Pharmacol 42:15691575[CrossRef][Medline]
-
Baker AR, McDonnell DP, Hughes MR, Crisp TM, Mangelsdorf
DJ, Haussler MR, Shine J, Pike JW, OMalley BW 1988 Molecular cloning
and expression of human vitamin D receptor complementary DNA:
structural homology with thyroid hormone receptor. Proc Natl Acad Sci
USA 85:32943298[Abstract]
-
Hughes MR, Malloy PJ, Kieback DG, Kesterson RA, Pike JW,
Feldman D, OMalley BW 1988 Point mutations in the human vitamin D
receptor gene associated with hypocalcemic rickets. Science 242:17021705[Medline]
-
Dilela AG, Woo SL 1987 Cloning large segments of genomic DNA
using cosmid vectors. Methods Enzymol 152:199212[Medline]
-
McDonnell DP, Scott RA, Kerner RA, OMalley BW, Pike JW 1989 Functional domains of the human vitamin D receptor regulate osteocalcin
gene expression. Mol Endocrinol 3:635644[Abstract]
-
Saijo T, Ito M, Takeda E, Huq AHM, Naito E, Yokoto I, Sone T,
Pike JW, Kuroda Y 1991 A unique mutation in vitamin D receptor gene in
three Japanese patients with vitamin D-dependent rickets type II:
utility of single-strand conformation polymorphism analysis for
heterozygous carrier detection. Am J Hum Genet 49:668673[Medline]
-
Dynan WS, Tjian R 1983 The promoter specific transcription
factor SP-1 binds to upstream sequences in the SV-40 early promoter.
Cell 35:7987[Medline]
-
Angel P, Imagawa M, Chiu R, Stein B, Imbra RJ, Rahmsdorf
JJ, Jonat C, Herrlich P, Karin M 1987 Phorbol ester inducible genes
contain a common cis element recognized by a TPA-modulated transacting
factor. Cell 49:729739[Medline]
-
Umesono K, Murakami KK, Thompson C, Evans RM 1991 Direct
repeats as selective response elements for the thyroid hormone,
retinoic acid, and vitamin D3 receptors. Cell 65:12551266[Medline]
-
Berg JM 1988 Proposed structure for the zinc binding domains
from transcription factor IIIA and related proteins. Proc Natl Acad Sci
USA 85:99102[Abstract]
-
Mader S, Kumar V, deVereneuil H, Chambon P 1989 Three
amino acids of the oestrogen receptor are essential to its ability to
distinquish an oestrogen from a glucocorticoid responsive receptor.
Nature 338:271274[CrossRef][Medline]
-
Hollenberg SM, Evans RM 1988 Multiple and cooperative
transactivation domains of the human glucocorticoid receptor. Cell 55:899906[Medline]
-
Tora L, White J, Brou C, Chambon P 1989 The human estrogen
receptor has two independent nonacidic transcriptional activation
functions. Cell 59:477487[Medline]
-
Tora L, Gronemeyer H, Turcotte B, Baub M-P, Chambon P 1988 The
N-terminal region of the chicken progesterone receptor specifies target
gene activation. Nature 333:185188[CrossRef][Medline]
-
van Leeuwen JPT M, Birkenhager JC, Vink-van Wijngaarden T,
van der Bemd GJC M, Pols HAP 1992 Regulation of 1,25-dihydroxyvitamin D
receptor gene expression by parathyroid hormone and cAMP agonists.
Biochem Biophys Res Commun 185:881886[Medline]
-
Van Leeuwen JPT M, Birkenhager JC, Buurman CJ, van der Bemd
GJC M, Pols HAP 1992 Bidirectional regulation of the
1,25-dihydroxyvitamin D receptor by phorbol ester activated protein
kinase C in osteoblast-like cells: interaction with adenosine
3',5'-monophosphate induced upregulation of the 1,25-dihydroxyvitamin D
receptor. Endocrinology 130:22592266[Abstract]
-
Kadonaga JT, Tjian R 1986 Affinity purification of sequence
specific DNA binding proteins. Proc Natl Acad Sci USA 83:58895893[Abstract]
-
Orkin SH 1995 Transcription factors and hematopoiesis. J
Biol Chem 270:49554958[Free Full Text]
-
Candeliere GA, Jurutka PW, Haussler MR, St. Arnaud R 1996 A
composite element binding the vitamin D receptor, retinoic X receptor
and a member of the CTF/NF-1 family of transcription factors
mediates the vitamin D responsiveness of the c-fos promoter. Mol Cell
Biol 16:584592[Abstract]
-
Gross C, Eccleshall TR, Mallow PJ, Villa ML, Marcus R, Feldman
D 1996 The presence of a polymorphism at the translation initiation
site of the vitamin D receptor gene is associated with low bone mineral
density in postmenopausal Mexican-American women. J Bone Miner Res 11:18501855[Medline]
-
Arai H, Miyamoto K, Taketani Y, Yamamoto H, Iemori Y, Morita
K, Tonai T, Nishisho T, Mori S, Takeda E 1997 A vitamin D receptor gene
polymorphism in the translation initiation codon: effect on protein
activity and relation to bone mineral density in Japanese women. J
Bone Miner Res, in press
-
Kamei Y, Kawada T, Fukuwatari T, Ono T, Kato S, Sugimoto E 1995 Cloning and sequence of the gene encoding the mouse vitamin D
receptor. Gene 152:281282[CrossRef][Medline]
-
Burmester JK, Maeda N, DeLuca HF 1988 Isolation and expression
of rat 1,25-dihydroxyvitamin D receptor cDNA. Proc Natl Acad Sci USA 85:10051009[Abstract]
-
Morrison NA, Qi JC, Tokita A, Kelly PJ, Crofts L, Nguyen TV,
Sambrook PN, Eisman JA 1994 Prediction of bone density from vitamin D
receptor alleles. Nature 367:284287[CrossRef][Medline]
-
Ozono K, Liao J, Kerner SA, Scott RA, Pike JW 1990 The vitamin
D responsive element in the human osteocalcin gene. J Biol Chem 265:2188121888[Abstract/Free Full Text]
-
Taylor JA, Hirvonen A, Watson M, Pittman G, Mohler JL, Bell DA 1996 Association of prostate cancer with vitamin D receptor gene
polymorphism. Cancer Res 56:41084110[Abstract]
-
Cooper GS, Umbach DM 1996 Are vitamin D receptor polymorphisms
associated with bone mineral density? A meta-analysis. J Bone
Miner Res 11:18411849[Medline]
-
Apte A, Siebert PD 1993 Anchor-ligated cDNA libraries: a
technique for generating a cDNA library for the immediate cloning of
the 5' ends of mRNAs. BioTechniques 15:890893[Medline]
-
Chomczynski P, Sacchi N 1987 Single step method of RNA
isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem 162:156159[CrossRef][Medline]
-
Gilman M 1987 Preparation and analysis of RNA. In: Ausubel FM,
Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds)
Current Protocols in Molecular Biology. John Wiley & Sons, New York, pp
4.7.14.7.8
-
Maniatis T, Fritch E, Sambrook J 1982 Molecular Cloning: A
Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY
-
Luckow B, Schutz G 1987 CAT constructions with multiple unique
restriction sites for the functional analysis of eukaryotic promoters
and regulatory elements. Nucleic Acids Res 15:5490[Medline]
-
Kawai S, Nishizawa M 1984 New procedures for DNA transfection
with polycation and dimethyl sulfoxide. Mol Cell Biol 4:11721174[Medline]
-
Nordeen SK 1988 Luciferase reporter gene vectors for analysis
of promoters and enhancers. Biotechniques 6:454457[Medline]
-
Tzukerman MT, Esty A, Santiso-Mere D, Danielian P, Parker MG,
Stein RB, Pike JW, McDonnell DP 1994 Human estrogen receptor
transactivational capacity is determined by both cellular and promoter
context and mediated by two functionally distinct intramolecular
regions. Mol Endocrinol 8:2130[Abstract]
-
Krust A, Green S, Argos P, Kumar V, Walter P, Bornert JM,
Chambon P 1986 The chicken oestrogen receptor sequence: homology with
v-erbA and the human oestrogen and glucocorticoid receptors. EMBO J 5:891897[Abstract]