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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
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-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
2–9) 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 |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 4–6 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).
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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. 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.
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-Phage Genomic Clones-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).
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-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.
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, 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.
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. 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 4–6 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 7–9 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.
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, pVDE1–1.93 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).
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, 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 |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-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 2–9) 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 VDR’s expression. A wealth of studies that describe the regulation of expression of the VDR provide the backdrop and rationale for these impending studies.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, 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 Denhardt’s
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 (pVDE1–3.2) as well as additional fragments were cloned into
the HindIII site of the luciferase expression vector pGL-2
basic (Promega). Plasmids pVDE1–2.6, pVDE1–1.93, pVDE1–1.48,
pVDE1–1.22, pVDE1–0.59, pVDE1–0.46, pVDE1–0.10, and pVDE1–0.03
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 Ham’s 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 16:584–592[Abstract]
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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. (
