Negative Regulation of the Androgen Receptor Gene Promoter by NFI and an Adjacently Located Multiprotein-Binding Site
Chung S. Song,
Myeong H. Jung1,
Prakash C. Supakar2,
Bandana Chatterjee and
Arun K. Roy
Department of Cellular and Structural Biology (C.S.S., M.H.J.,
P.C.S., B.C., A.K.R.) The University of Texas Health Science Center
at San Antonio San Antonio, Texas 78284
Audie L.
Murphy Memorial VA Hospital (B.C.) San Antonio, Texas 78284
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ABSTRACT
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The upstream promoter of the rat androgen receptor
(AR) gene contains a strong negative regulatory region located at the
-388 to -340 nucleotide position. The distal part (-388/-373) of
this regulatory region binds NFI, a ubiquitous transcription factor,
while the proximal portion (-372/-340) contains an overlapping
binding site for two nuclear proteins. This composite regulatory region
(-388/-340) was initially defined by deoxyribonuclease I footprinting
as the continuous stretch of a nuclease-protected site. NFI specificity
of the distal portion (-388/-373) of the footprint was established
through cross-competition in electrophoretic mobility shift assay
(EMSA) using the well characterized NFI element of the adenovirus major
late promoter and by immunoreactivity to the NFI antibody. EMSA with
oligonucleotide duplexes corresponding to the proximal domain
(-372/-340) indicated multiple retarded bands with at least two major
DNA-protein complexes. Further analysis with truncated oligonucleotide
duplexes showed that these two major proteins bind to this domain in an
overlapping manner. Within this overlapping area, the position spanning
-359 to -347 is essential for the formation of either of these two
complexes. Substitution of four G with T residues in the overlapping
area totally abolished all protein binding at the downstream
-372/-340 site. Point mutations that abolish specific binding at
either the NFI or immediately downstream multiprotein-binding site
caused about a 10-fold increase in AR promoter activity in transfected
HepG2 cells. Double mutation involving both the NFI and proximal
overlapping protein-binding sites failed to cause any additional
increase in promoter function. From these results we conclude that the
AR promoter contains a composite negative regulatory region at
-388/-340, and the repressor function may involve a coordinate
interaction between NFI and at least two other nuclear factors.
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INTRODUCTION
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Androgenic steroids are critical signaling agents for the
development and regulation of the male sexual phenotype (1). Similar to
other steroid hormones, androgens influence gene expression by binding
and activating the androgen receptor (AR) protein and by recruiting
other accessory coactivators at target genes (2). Intracellular
concentrations of both the receptor and the receptor-activating steroid
ligand appear to play almost equally important roles in the mediation
of androgen action (3). Cell-specific expression of the AR gene during
development, maturation, and aging can determine the spatiotemporal
control of target cell sensitivity to androgenic hormones. Both
negative and positive controls of the AR promoter function are
responsible for these tissue- and age-dependent differences in hormonal
sensitivity. Understanding the role of various trans-acting proteins in
the regulation of the AR promoter is essential to elucidate the
mechanism of the spatiotemporal changes in androgen action. Studies in
our laboratory and by others have helped to characterize the AR gene
promoters from a number of species including mouse, rat, dog, and human
(4, 5, 6, 7, 8, 9, 10, 11, 12). In addition to the extensive sequence homology among different
mammalian species, the salient features of the AR gene promoter are the
absence of a TATA- or CAAT-box near the initiation site, the presence
of an approximately 100-bp long homopurine/homopyrimidine (pur/pyr)
element immediately upstream of the Sp1-box, and a number of specific
transcription factor-binding sites further upstream. A comparative
analysis of the AR promoter sequences through phylogenetic footprinting
(13) suggests the presence of about 20 transcription factor-binding
sites between -1000 and -150 bp (3, 14). Since the AR is expressed
only at a very low level in most tissues, except reproductive organs,
many of these potential protein-binding sites may serve to
quantitatively lower its rate of transcription in nonreproductive
tissues by interacting with negative regulators. In this report we
describe the identification of a strong negative regulatory element at
the AR gene promoter containing NFI and at least two other nuclear
factors.
NFI represents a family of enhancer binding proteins originally
identified as a host initiation factor for adenoviral DNA replication
(15). In vertebrate species, four closely related isoforms of NFI are
coded by separate genes, NFI-A, NFI-B, NFI-C, and NFI-X. Primary
transcripts of these isoform-specific genes also undergo alternate
splicing, generating a family of transcription factors (16). Specific
DNA binding sites for NFI and its positive regulatory role in the
transcriptional control of a large number of eukaryotic genes have been
established (17, 18, 19, 20). In addition to its predominantly positive
regulatory function, in a number of cases negative regulatory effects
of NFI have also been described. These include retinol-binding protein
(21),
2(I) collagen (22), osteonectin (23), lipoprotein lipase (24),
GH (25), neuron-specific peripherin in nonneuronal cells (26), von
Willebrand factor (27), glutathione transferase P (28), and PIT-1 (29).
Unlike its positive regulatory function, which is mediated through
interactions with the basal transcriptional machinery, the mechanism of
the negative regulatory function of NFI is largely unknown.
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RESULTS
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Deoxyribonuclease I (DNase I) footprinting of the rat AR promoter
shows a long stretch of protected region covering -388 to -340 bp
(Fig. 1
). A transcription factor database
search revealed that the upstream portion (-388 to -373) of the
footprint corresponds to the binding sequence of the transcription
factor NFI (18). However, the rest of the protected region (-372 to
-340) does not show homology to the consensus binding motif for any
known DNA-binding protein. Despite the continuous nature of the
protected region, the upstream and downstream components of the
footprint competed distinctively for separate binding proteins.
Selective competition of these two footprint positions, either by a
33-mer oligonucleotide duplex representing the proximal segment of the
footprinted DNA or a 30-mer duplex representing the high-affinity NFI
enhancer element of the adenovirus major late promoter, is shown in
lanes 3 and 4, respectively (Fig. 1
).

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Figure 1. DNase I Footprint Pattern of the Rat AR Gene
Promoter Spanning -486 to -324 bp Positions
Lanes 1 and 5, Naked DNA digested with DNase I; lane 2, DNA treated
with rat liver nuclear extract before digestion with DNase I; lane 3,
same as 2 except with a 300-fold molar excess of a competitor
oligonucleotide duplex corresponding to -372 to -340 positions of the
rat AR gene; lane 4, same as 2 except with a 300-fold molar excess of a
competitor oligonucleotide duplex corresponding to the NFI element of
the adenovirus major late (AdML) promoter. Numbers on the
left show base positions within the promoter sequence. The
nucleotide sequences of the footprint, the putative NFI element, and
the MBS are shown on the right.
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Identity of the -388/-373 footprint site as the NFI element was
substantiated by cross-competition between the AR promoter sequence and
the adenoviral NFI element in electrophoretic mobility shift assay
(EMSA) (Fig. 2
). The labeled
oligonucleotide duplex corresponding to the -388/-373 footprint
produced a retarded complex containing closely migrating bands
characteristic of the EMSA pattern produced by the multiple forms of
the NFI protein. These closely migrating bands can be almost completely
competed out with either a 50-fold molar excess of the unlabeled
homologous oligonucleotide (lane 4), or 10-fold molar excess of a
30-mer duplex corresponding to the NFI-binding site of the adenovirus
major late (AdML) promoter (lane 5). However, even 100-fold molar
excess of another closely related cis-element cognate to
CCAAT/enhancer-binding protein (C/EBP) (30, 31) failed to cause
any significant reduction of the band intensity (lane 8).

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Figure 2. Electrophoretic Mobility Shift Pattern of the Rat
AR -388/-373 Element
32P-labeled oligonucleotide duplex corresponding to the
-388/-373 rat AR element was used as the probe in mobility shift
assay. Competition of the specific protein binding by different
oligonucleotide duplexes and cis-elements at indicated
fold molar excesses are shown on the top.
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Identity of the bound proteins as NFI isoforms was further
authenticated by their immunoreactivity with the polyclonal antibody to
NFI (Fig. 3
, lane 6). The adenovirus NFI
element contains two CCAAT-like boxes (box-A and -B) that are present
in an inverted orientation, while in the AR promoter these two boxes
occur in a direct orientation (Table 1
).
Point mutations at three of the five bases within the B-box of the
-388/-373 footprinted site of the AR promoter prevented it from
functioning as an effective competitor for the wild-type AR element
(Fig. 3
, lane 4). From all of these results we can conclude that the AR
-388/-373 footprint site contains an authentic NFI element, but its
binding affinity is lower than that of the adenovirus NFI
site.

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Figure 3. Mutational Inactivation and Immunoreactivity of the
Rat AR NFI Binding Site
Lanes 13 show band patterns without any competitor oligonucleotide,
with 100-fold molar excesses of the homologous rat AR -388/-373
oligo, and the adenovirus major late promoter NFI oligo, respectively.
Lane 4 shows lack of competition with the mutant -388/-373 oligo
containing three- point mutations (described in Materials and
Methods). Lane 5 shows lack of competition with the related
cis-element for C/EBP. Lanes 6 and 7 show that
polyclonal antibody to NFI, but not the preimmune IgG, eliminates
specific protein binding.
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Table 1. Sequence Comparison between -388/-373 Site
and the NF1-Binding Site of the Adenovirus Major Late Promoter
(AdMLP)
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The proximal part (-372/-340) of the footprinted site appears
to contain binding sites for at least two uncharacterized DNA-binding
proteins. EMSA with a labeled probe corresponding to this footprinted
site and the rat liver nuclear extract suggested multiple protein
binding with a distinct slower-migrating upper complex and a
faster-migrating lower complex (Fig. 4
).
Although protein binding to both of these complexes can be competed out
with a 100-fold excess of unlabeled homologous oligonucleotide duplex
(lane 2), oligonucleotides corresponding to the consensus binding
elements of two other transcription factors, C/EBP and NF
B, at the
same molar ratio failed to compete for binding with the labeled probe
(lanes 3 and 4). These results and our failure to find any match of
sequence homology from the transcription factor database have led us to
conclude that the rat AR -372/-340 site specifically binds to at
least two yet-to-be-characterized sequence-specific DNA-binding
proteins.

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Figure 4. Multiple Protein Binding at the -372/-340 Rat AR-
Regulatory Element
Electrophoretic mobility shift with the 32P-labeled
-372/-340 oligonucleotide duplex was performed with the rat liver
nuclear extract. Competition of the specific binding with 100-fold
molar excesses of the unlabeled homologous oligo, NF B consensus
element, and C/EBP consensus element are shown in lanes 2, 3, and 4,
respectively.
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To further delineate this multiprotein binding site (MBS), EMSA with
labeled probes containing truncated sequences was performed. As shown
in Fig. 5
, removal of 5 bp from the
3'-end is sufficient to cause a marked decline in the formation of both
the lower and the upper complexes, and removal of 2 more base pairs
from the 3'-end almost completely disrupted both of these complexes.
Removal of 8 bp from the 5'-boundary of the proximal half of the
footprint site did not disrupt formation of the upper complex but
generated a new complex of intermediate mobility (lane 4). However,
truncation of 11 bp totally eliminated the upper complex and at the
same time increased the intensity of the intermediate complex (lane 5).
Removal of 13 bp from the 5'-end abolished all protein binding to the
DNA duplex (lane 6), and no specifically retarded bands were observed.
From all of these results, we conclude that the -372/-340 segment of
the footprinted site contains overlapping sites for multiple
DNA-binding proteins, and that the DNA sequence encompassing -347 to
-359 bp is essential for the formation of all of these DNA-protein
complexes. This essential component (-347/-359) contains four G
residues on the upper strand, and mutation of these G residues to T
completely abolished the ability of the -372/-340 oligonucleotide
duplex to bind to cognate binding proteins (Fig. 5
, lane 7).

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Figure 5. The Overlapping Nature of Specific Protein Binding
at the -372/-340 Rat AR-Regulatory Element
Each lane in the upper panel (A) shows the
electrophoretic mobility shift pattern of individually labeled
oligonucleotide duplexes as indicated on the top. The
lower panel (B) shows the nucleotide sequences of the
oligonucleotides used for EMSA in panel A. The central core region
(-359/-347) essential for formation of both of the retarded complexes
is indicated with a box at the top, and mutant positions
are marked with underlines at the bottom.
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The functional role of the composite protein-binding element covering
the entire footprinted region (-388/-340) was examined by
transfectional analysis of promoter-reporter constructs containing a
promoter fragment spanning -1040 to +560 bp of the rat AR gene ligated
to the firefly luciferase coding DNA. A mutated form of the proximal
MBS (mMBS) of the promoter-reporter construct contained four-base
substitutions (GGCCTGTG
TTCCTTTT) within the essential
-347/-359 binding region. The promoter mutated at the NFI site (mNFI)
contained substitutions at sequences from -378 to -376 (TGG
GTT).
In EMSA, both of these mutations were shown to destroy specific binding
activity to cognate binding proteins (Fig. 3
, lane 4, and Fig. 5
, lane
7). In addition to the promoter-reporter constructs that contain
individual mutations at these two sites, a third double mutant (DMT)
containing mutations within both of these protein-binding sites was
also tested in cell transfection assay. Results presented in Fig. 6
show that inactivation of protein
binding by mutations at either the NFI site (-388/-373) or the
proximal multiprotein-binding site (-372/-340) caused an
approximately 10-fold increase in the promoter activity. However,
mutations at both of these sites together (DMT) did not produce any
additional increase in the promoter activity. These results indicate
that the entire footprinted area spanning -388 to -340 functions as a
negative regulatory region of the rat AR gene, and the same degree of
derepression after mutational inactivation of either one or both of
these sites suggests a cooperative interaction among these
sequence-specific DNA-binding proteins.

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Figure 6. Relative Activities of the Wild-Type and Mutant
Forms of the AR Promoter in Transfected HepG2 Cells
WT, wild type; mMBS, mutated MBS; mNFI, mutated NFI site; DMT, a double
mutant containing mutations at both the MBS and the NFI site. Each
point is derived from separate transfection experiments with
corresponding wild-type controls.
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NFI appears to be expressed ubiquitously, albeit in a cell- and
tissue-specific isoform composition. We have examined the cellular
distribution of NFI and the unknown nuclear factors that interact with
the proximal portion of the footprinted sequence in nuclear extracts
derived from cells of different tissue origins (Fig. 7
). The isoform composition of NFI in
nuclear extracts derived from the liver and kidney (as indicated by the
gel shift pattern) showed a marked difference, with the kidney extract
displaying a predominance of higher mobility complexes (Fig. 7A
, lanes
1 and 2). However, predominance of NFI isoforms that yield higher
mobility DNA-protein complexes does not appear to correlate with a
higher level of AR expression. This is indicated by band patterns shown
in the next two lanes (lanes 3 and 4), where the AR-positive LNCaP
cell extract shows slower migrating bands as compared with the
AR-negative PC3 cells. Nuclear extracts from the other four cell lines,
i.e. CHO, HeLa, COS1, and FTO2B (lanes 58), show band
patterns similar to those of the rat liver (lane 1). The component
binding proteins for the proximal (-372/-340) portion of the
regulatory region appear to be present not only in the rat liver but
also in nuclear extracts derived from the kidney and from LNCaP, PC3,
CHO, HeLa, COS1, and FTO2B cells (Fig. 7B
). Although tissue and
cellular distributions of the ratio of the two component bands show
some variations, no correlation of band patterns between AR-expressing
tissues and cell lines (liver, kidney, and LNCaP) and AR-negative cells
was observed. All tissue and cell extracts that were examined contained
both of these specific DNA-binding proteins. These results suggest that
derepression of the AR gene from the negative control of this composite
regulatory element may not be due to simple tissue-specific differences
in levels of the nuclear factors that specifically bind to this
promoter region, and may, in fact, be the result of complex interaction
of this element with other regulatory regions in the context of
nucleosomal structure and/or interactions with other proteins that can
function as corepressors along with these sequence-specific
enhancer-binding factors.

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Figure 7. Ubiquitous Expression of NFI and the Nuclear
Proteins That Bind to the -372/-340 Site of the Rat AR Gene
Various nuclear extracts used for the EMSA are indicated at the
top. A, EMSA with the labeled NFI (-388/-373) probe;
B, EMSA with the adjacent multiprotein binding site (-372/-340)
labeled probe.
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DISCUSSION
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The AR gene is expressed in all tissues of the rat, albeit at
markedly different levels. A relatively high level of AR expression is
found only in specific cells of the male reproductive tissues and in
the adrenal cortex and kidney (3). Even where it is expressed at a low
level, as in the liver, the level of expression can vary over a range
of approximately 100-fold at different stages of life (7). Efficient
tissue-specific expression of the AR at a low level and its regulation
during maturation and aging require interactions among a number of
positive (7, 8, 10, 12) and negative regulatory elements (9, 11, 12).
In several earlier reports we have described the positive regulatory
role of an age-dependent factor and Sp1 in rat AR gene
expression and negative regulation of this receptor gene by NF
B, all
of which undergo marked age-dependent changes (7, 11, 12). Evidence for
the presence of a strong negative control region presented in this
article further extends the regulatory mechanisms available for
spatiotemporal expression of the AR gene.
The ubiquitous transcription factor NFI appears to be one of the
important regulatory components of the multiprotein binding site at
-388/-340 of the rat AR promoter. The NFI family of transcription
factors primarily functions as a positive regulator; nevertheless, in
addition to the present report, the negative regulatory role of NFI has
also been described for a number of genes (21, 22, 23, 24, 25, 26, 27, 28, 29). Unlike its positive
regulatory function, which is mediated by direct interaction of the
enhancer-bound NFI to the general transcription factors, the
mechanism(s) for the NFI-mediated negative regulation is still largely
unknown. Gil et al. (19) initially proposed that the
sterol-mediated feedback inhibition of HMG-CoA reductase gene
expression may be mediated through disruption of the protein-protein
interaction between NFI and a hypothetical downstream partner. The
presence of such downstream protein-binding sites within the negative
regulatory elements of both the rat AR gene and the rat GH gene
provides experimental support for such a hypothetical mechanism. The
structural arrangement and regulatory properties of the NFI-dependent
negative regulatory region of the rat GH gene (also known as the
silencer-1) are very similar to the negative regulatory region of the
rat AR as described in this article. The silencer-1 region of the rat
GH gene contains an NFI-binding site that is immediately followed
downstream by the binding site of a yet-to-be-characterized nuclear
protein called SBP2 (silencer binding protein 2) (32). Although the
SBP2 site does not have any sequence homology to the MBS site of the
rat AR gene, analogous to the case for the rat AR promoter, both NFI
and SBP2 elements of the rat GH promoter provide independent, but not
additive, repressive effects, indicating a cooperative mode of function
between these two regulatory sites.
In an attempt to identify the presence of any specific repression
domain within the NFI protein sequence, Osada et al. (33)
have tested the effects of various N-terminal truncated forms of NFIA
(the predominant NFI isoform of the rat liver) ligated to the
heterologous yeast GAL4 DNA-binding domain on the negative regulation
of the rat glutathione transferase P gene promoter. From these studies
they concluded that the minimum repression domain is contained within
the 318427 amino acid region that is conserved in all four major
isoforms of the NFI protein. However,
glutathione-S-transferase pull-down assay failed to
identify interaction of this domain with any one of the general
transcription factor components of the preinitiation complex. Despite
these results showing a lack of interaction between the negative
regulatory domain of NFI with the known members of the preinitiation
complex, the possibility that non-DNA-binding corepressors may
contribute to the repressor function of NFI still remains to be
examined. Recently, Gao and Kunos (34) have reported that
overexpression of NFI in transfected Hep3B cells results in the
activation of the
1B adrenergic receptor gene middle
promoter, whereas in another cell type DDT MF-2, the same NFI isoform
acts as a negative regulator of promoter function. Results of these
studies also suggest that a cell type-specific expression of a non-DNA-
binding nuclear protein may modulate the repressor function of NFI-X
(the specific isoform used in their study) through protein-protein
interaction at a region located between 243 and 416 amino acid
positions of NFI-X. Although in our case we did not observe any major
tissue-specific differences in the level of the two adjacently
positioned DNA-binding proteins, all of these published results point
to more than one mechanisms for the negative regulatory function of the
NFI proteins.
With the exception of the major androgen targets such as the prostate,
seminal vesicle, testis, adrenal cortex, and kidney, in most tissues
the AR mRNA is expressed at a low level, but in a highly controlled
fashion (3). Negative regulatory mechanisms appear to play major roles
in such muted expression of the AR gene, specifically in
nonreproductive tissues. In addition to its AR-mediated autoregulation
(35, 36, 37, 38, 39, 40), which may have a greater importance in tissues where AR is
expressed at a relatively abundant level, several negatively acting
cis-elements have been described. These include two negative
regulatory regions in the mouse AR with yet-to-be-characterized binding
proteins (9, 41), the NF-
B element (11), and the single-strand
pyrimidine-specific protein-binding site in the rat AR promoter (12).
Among all of these regulatory elements, the NFI composite element
described in this paper appears to provide the strongest repressor
function in transient transfections. Unlike the marked increase in
NF-
B during the age-dependent down-regulation of AR in the rat
liver, we have not observed any correlation of the hepatic levels of AR
mRNAs at different ages with the nuclear levels of NFI or the
adjacently located DNA-binding proteins. Although the results presented
in this article do not show any correlation between the
isoform-dependent gel shift patterns of NFI and AR expression in
different cell types, a significant downward shift in the
electrophoretic mobility of NFI-DNA complexes in the nuclear extracts
derived from old rats has been reported (42). Whether such differences
in the NFI isoform composition, generated either by alternate splicing
of the primary transcript (31) or by O-linked glycosylation of the
protein (43), play any significant role in the age-dependent
down-regulation of AR in the rat liver remains uncertain. However,
altered expression of C/EBP isoforms during aging has been implicated
in the changes in hepatic gene expression (44).
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MATERIALS AND METHODS
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Preparation of Nuclear Extracts
Fischer 344 male rats (34 months of age) were obtained from
Charles River Laboratories, Inc., Wilmington, MA. All
animal experiments were conducted in accordance with NIH standards of
humane care, and all protocols were approved by the Institutional
Animal Care Committee. Nuclear extracts from rat liver and kidney were
prepared according to the method described by Hattori et al.
(45). Nuclear extracts from HeLa, FTO2B, PC3, LNCaP, CHO, and COS1
cells were prepared by the method of Dignam et al. (46). All
buffers contained 2 µg/ml each of aprotinin, leupeptin, bestatin, 0.1
mM phenylmethyl sulfonyl fluoride, and 1 mM
dithiothreitol (DTT). Protein concentrations were determined using the
Bradford assay.
EMSA
Oligonucleotide probes were labeled with 32P using
T4 polynucleotide kinase and [
-32P]-ATP. Five
micrograms of nuclear extracts were preincubated with 2 µg of
poly(dl-dC) for 5 min in 10 mM Tris-HCl, pH 7.5, 50
mM NaCl, 1 mM DTT, 1 mM EDTA, 10%
glycerol at room temperature with or without the unlabeled competitor
DNA. After preincubation, the labeled probe (1020 fmol) was added to
the reaction mixture and incubated for 20 min. Protein-DNA complexes
were resolved by electrophoresis on 5% polyacrylamide gels. After
electrophoresis, gels were dried and autoradiographed. The antibody
supershift experiments were carried out using 5 µl polyclonal rabbit
antiserum to NF1 containing 5 µg of IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The antibody was
preincubated with the nuclear extract for 45 min at 22 C. The reaction
mixture was then incubated with poly(dl-dC) for 5 min before the
addition of the radiolabeled probe, and incubation continued for an
additional 20 min. For the control experiment, IgG from the preimmune
rabbit serum was used in place of the NF1 antibody. DNA-protein
complexes were analyzed by EMSA.
DNase I Footprinting
DNase I footprinting of the rat AR promoter was performed
according to the procedure described previously (47). Briefly, the 3'
end-radiolabeled DNA fragment was generated by PCR using
32P-radiolabeled antisense primer (-280/-300) and
unlabeled sense primer (-665/-645). The radiolabeled probe (50,000
cpm, 10 fmol) was incubated with 50 µg of nuclear extract in a 50
µl reaction mixture at 10 mM HEPES, pH 7.6, 60
mM KCl, 5% (vol/vol) glycerol, 0.5 mM DTT, 0.5
mM EDTA, and 2 µg of poly(dI-dC) double-stranded DNA.
After preincubation of the reaction mixture (10 min, room temperature)
without the DNA probe, the labeled DNA was added and incubation
continued on ice for 30 min. The reaction mixture was then brought to 1
mM CaCl2 and MgCl2 and incubated at
room temperature for 1 min, and then the protein-bound DNA was digested
with 0.020.1 µg of DNase I (30 sec to 2 min, room temperature)
under standard buffer conditions (47). For incubations with BSA,
10-fold less DNase I was used. Digested DNA fragments were extracted
with phenol-chloroform (1:1), resolved on an 8% sequencing gel, and
visualized by autoradiography.
Construction of Wild-Type and Mutant Plasmids
The wild-type plasmid (pAR 1.6 kb-Luc) used in this study
contains the wild-type rat AR promoter from -1040 to +560 bp inserted
into the luciferase vector pGL2 Basic (Promega Corp.). The
four-point mutant at the proximal MBS (mMBS) contains four-base
substitutions (GGCCTGTG
TTCCTTTT) within the core sequence
(-347/-359). The three-point mutant at the distal NF1 binding site
contains three-base substitutions within the footprinted site spanning
-378 to -376 (TGG
GTT). The site-specific
mutations were introduced into DNA fragments generated by PCR of the
wild-type pAR 1.6-kb Luc plasmid template, in the presence of the
mutant oligonucleotide primer containing the appropriate base
substitutions and the vector-specific primer. The mutant DNA fragments
generated by PCR were digested with restriction enzymes and purified by
gel electrophoresis. DNA fragments containing the desired mutations
were then reinserted into the wild-type plasmid sequence and
authenticated by DNA sequencing.
Analysis of Promoter Function in Transfected Cells
The strength of the AR promoter to direct luciferase expression
was measured in transfected HepG2 (human hepatoma) cells as described
earlier (11, 12). Briefly, approximately 106 cells were
seeded in the T25 flask. After overnight culture in
DMEM-Hanks F12 medium (1:1) containing 10% FBS, cells were
transfected with the plasmid DNA following the calcium phosphate
coprecipitation method. After transfection, cells were washed with PBS
(pH 7.5), subjected to glycerol shock (10%, 3 min), and cultured for
an additional 48 h before harvesting. Cytoplasmic extracts were
assayed for both luciferase activity (48) and protein concentration,
and the enzyme activities in different samples were normalized to the
constant amount of the total protein.
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ACKNOWLEDGMENTS
|
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We thank Tina Hassan, Sang Kim, and Gilbert Torralva for
dedicated technical assistance and Lita Chambers for secretarial help.
Mutant promoter-reporter constructs used in this study were prepared by
Sun-Jin Choi.
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FOOTNOTES
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Address requests for reprints to: Dr. Arun K. Roy, Department of Cellular and Structural Biology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7762.
This work was supported by NIH Grants, AG-10486 and DK-14744. B.C. is a
career scientist with the Department of Veterans Affairs.
1 Current address: National Institute of Health, Division of Cancer
Research, Seoul 122020, Korea. 
2 Current address: Institute of Life Sciences, Bhubaneswar 751007,
India. 
Received for publication April 12, 1999.
Revision received June 11, 1999.
Accepted for publication June 21, 1999.
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REFERENCES
|
---|
-
Wilson JD, Griffin JE, Russell DW 1993 Steroid 5
-reductase 2 deficiency. Endocr Rev 14:577593[Abstract]
-
Kokontis J, Liao S 1999 Molecular action of androgen in the
normal and neoplastic prostate. Vitam Horm 55:220307
-
Roy AK, Lavrovsky Y, Song CS, Chen S, Jung MH, Velu NK, Bi
BY, Chatterjee B 1999 Regulation of androgen action. Vitam Horm 55:309352[Medline]
-
Tilley WD, Marcelli M, McPhaul MJ 1990 Expression of the
human androgen receptor gene utilizes a common promoter in diverse
human tissues and cell lines. J Biol Chem 265:1377613781[Abstract/Free Full Text]
-
Baarends WM, Themmen APN, Blok LJ, Mackenbach P, Brinkmann
AO, Meijer D, Faber PW, Trapman J, Grootegoed JA 1990 The rat androgen
receptor gene promoter. Mol Cell Endocrinol 74:7584[CrossRef][Medline]
-
Song CS, Her S, Choi SJ, Slomczynska M, Roy AK, Chatterjee B 1993 A distal activation domain is critical in the regulation of the
rat androgen receptor gene promoter. Biochem J 294:779784[Medline]
-
Supakar PC, Song CS, Jung MH, Slomczynska MA, Kim JM,
Vellanoweth RL, Chatterjee B, Roy AK 1993 A novel regulatory element
associated with age-dependent expression of the rat androgen receptor
gene. J Biol Chem 268:2640026408[Abstract/Free Full Text]
-
Faber PW, van Rooij JCJ, Schipper HJ, Brinkmann AO, Trapman J 1993 Two different, overlapping pathways of transcription initiation
are active on the TATA-less human androgen receptor promoter. The role
of Sp 1. J Biol Chem 268:92969301[Abstract/Free Full Text]
-
Kumar MV, Jones EA, Grossmann ME, Blexrud MD, Tindall DJ 1994 Identification and characterization of a suppressor element in the
5'-flanking region of the mouse androgen receptor gene. Nucleic Acids
Res 22:36933698[Abstract]
-
Mizokami A, Yeh SY, Chang C 1994 Identification of 3',
5'-cyclic adenosine monophosphate response element and other cis-acting
elements in the human androgen receptor gene promoter. Mol Endocrinol 8:7788[Abstract]
-
Supakar PC, Jung MH, Song CS, Chatterjee B, Roy AK 1995 Nuclear factor kappa B functions as a negative regulator for the rat
androgen receptor gene and NF-kappa B activity increases during the
age-dependent desensitization of the liver. J Biol Chem 270:837842[Abstract/Free Full Text]
-
Chen S, Supakar PC, Vellanoweth RL, Song CS, Chatterjee B, Roy
AK 1997 Functional role of a conformationally flexible
homopurine/homopyrimidine domain of the androgen receptor gene promoter
interacting with Sp 1 and a pyrimidine single strand DNA-binding
protein. Mol Endocrinol 11:315[Abstract/Free Full Text]
-
Gumucio DL, Shelton DA, Bailey WJ, Slightom JL, Goodman M 1993 Phylogenetic footprinting reveals unexpected complexity in trans factor
binding upstream from the
-globin gene. Proc Natl Acad Sci USA 90:60186022[Abstract]
-
Roy AK, Chatterjee B 1995 Androgen action. Crit Rev Euk Gene
Expr 5:157176[Medline]
-
Gronostajski RM, Adhya S, Nagata K, Guggenheimer RA, Hurwitz J 1985 Site-specific DNA binding of nuclear factor I: analyses of
cellular binding sites. Mol Cell Biol 5:964971[Medline]
-
Kruse U, Sippel AE 1994 The genes for transcription factor
nuclear factor I give rise to corresponding splice variants between
vertebrate species. J Mol Biol 238:860865[CrossRef][Medline]
-
Jones KA, Kadonaga JT, Rosenfeld PJ, Kelly TJ, Tjian R 1987 A
cellular DNA-binding protein that activates eukaryotic transcription
and DNA replication. Cell 48:7989[Medline]
-
Chodosh LA, Baldwin AS, Carthew RW, Sharp PA 1988 Human CCA
AT-binding proteins have heterologous subunits. Cell 53:1124[Medline]
-
Gil G, Smith JR, Goldstein JL, Slaughter CA, Orth K, Brown MS,
Osborne TF 1988 Multiple genes encode nuclear factor 1-like proteins
that bind to the promoter for 3-hydroxy-3-methylglutary1-coenzyme A
reductase. Proc Natl Acad Sci USA 85:89638967[Abstract]
-
Paonessa G, Gourani F, Frank R, Cortese R 1988 Purification of
a NF1-like DNA-binding protein from rat liver and cloning of the
corresponding c DNA. EMBO J 7:31153123[Abstract]
-
Colantuoni V, Pirrozi A, Blance C, Cortese R 1987 Negative
control of liver-specific gene expression: cloned human retinol-binding
protein gene is repressed in HeLa cellls. EMBO J 6:631636[Abstract]
-
Rossi P, Karsenty G, Roberts AB, Roche NS, Sporn M, de
Crombrugghe B 1988 A nuclear factor 1 binding site mediates the
transcriptional activation of a type I collagen promoter by
transforming growth factor-ß. Cell 52:405414[Medline]
-
Nomura S, Hashmi S, McVey JJ, Ham J, Parker M, Hogan B 1989 Evidence for positive and negative regulatory elements in the
5'-flanking sequence of the mouse sparc (osteonectin) gene. J Biol
Chem 264:1220112207[Abstract/Free Full Text]
-
Schoonjans K, Staels B, Devos P, Szpirer J, Szpirer C, Deeb S,
Verhoeven G, Auwerx J 1993 Development extinction of liver lipoprotein
lipase m RNA expression might be regulated by an NF-1-like site. FEBS
Lett 329:8995[CrossRef][Medline]
-
Roy RJ, Vallieres L, Leclerc S, Guerin SL 1994 The rat growth
hormone proximal silencer contains a novel DNA-binding site for
multiple nuclear protein that represses basal promoter activity. Eur
J Biochem 225:419432[Abstract]
-
Adams AD, Choate DM, Thompson MA 1995 NF1-L is the DNA-binding
component of the protein complex at the peripherin negative regulatory
element. J Biol Chem 270:69756983[Abstract/Free Full Text]
-
Jahroudi N, Ardekani AM, Greenberger JS 1996 An NF1-like
protein functions as a repressor of the von Willebrand factor promoter.
J Biol Chem 271:2141321421[Abstract/Free Full Text]
-
Osada S, Daimon S, Ikeda T, Nishihara T, Yano K, Yamasaki M,
Imagawa M (1997) Nuclear factor 1 family proteins bind to the ssilencer
element in the rat glutathione transferase P gene. J Biochem 121:356363
-
Rajas F, Delhase M, de la Hoya M, Verdood P, Castrillo J,
Hooghe-Peters EL (1998) Nuclear factor 1 regulates the distal silencer
of the human PIT1/GHF1 gene. Biochem J 333:7784
-
Christy RJ, Yang VW, Ntambi JM, Geiman DE, Landschulz WH,
Friedman AD, Nakabeppu Y, Kelly TJ, Lane MD 1989 Differentiation-induced gene expression in 3T3-L 1 preadipocytes:
CCAAT/enhancer binding protein interacts with and activates the
promoter of two adipocyte-specific genes. Genes Dev 3:13231335[Abstract]
-
Santoro C, Mermod N, Andrews PC, Tjian R 1988 A family of
human CCA AT-box-binding proteins active in transcription and DNA
replication: cloning and expression of multiple cDNAs. Nature 334:218224[CrossRef][Medline]
-
Roy RJ, Guerin SL 1993 Two distinct nuclear proteins bind to
the rat growth hormone silencer-1 element. Ann NY Acad Sci 684:207210[Medline]
-
Osada S, Ikeda T, Xu M, Nishihara T, Imagawa M 1997 Identification of an extended half-site motif required for the function
of peroxisome proliferator-activated receptor
. Biochem Biophys Res
Commun 238:744747[CrossRef][Medline]
-
Gao B, Kunos G 1998 Cell type-specific transcriptional
activation and suppression of the
1 B adrenergic receptor gene
middle promoter by nuclear factor 1. J Biol Chem 273:3178431787[Abstract/Free Full Text]
-
Krongrad A, Wilson CM, Wilson JD, Allman DR, McPhaul MJ 1991 Androgen increases androgen receptor protein while decreasing receptor
mRNA in LN CaP cells. Mol Cell Endocrinol 76:7988[CrossRef][Medline]
-
Wolf DA, Herzinger T, Hermeking H, Blaschke D, Horz W 1993 Transcriptional and posttranscriptional regulation of human androgen
receptor expression by androgen. Mol Endocrinol 7:924936[Abstract]
-
Grossman ME, Lindzey J, Blok L, Perry JE, Kumar MV,
Tindall DJ 1994 The mouse androgen receptor gene contains a second
functional promoter which is regulated by dihydrotestosterone.
Biochemistry 33:1459414600[Medline]
-
Mora GR, Mahesh VB 1996 Autoregulation of androgen receptor in
rat ventral prostate: involvement of c-fos as a negative
regulator. Mol Cell Endocrinol 124:111120[CrossRef][Medline]
-
Dai JL, Burnstein KL 1996 Two androgen rsponse elements inthe
androgen receptor coding region are required for cell-specific
up-regulation of receptor messenger RNA. Mol Endocrinol 10:15821594[Abstract]
-
Wiren KM, Zhang X, Chang C, Keenan E, Orwoll ES 1997 Transcriptional up-regulation of the human androgen receptor by
androgen in bone cells. Endocrinology 138:22912300[Abstract/Free Full Text]
-
Grossman ME, Tindall DJ 1995 The androgen receptor is
transcriptionally suppressed by proteins that bind single-stranded DNA.
J Biol Chem 270:1096810975[Abstract/Free Full Text]
-
Supakar PC, Roy AK 1996 Role of transcription factors in the
age-dependent regulation of the androgen receptor gene in rat liver.
Biol Signals 5:170179[Medline]
-
Jackson SP, Tjian R 1988 O-glycosylation of eukaryotic
transcription factors: implication for mechanisms of transcriptional
regulation. Cell 55:125133[Medline]
-
Hsieh CC, Xiong W, Xie Q, Rabek JP, Scott SG, An MR, Reisner
PD, Kuninger DT, Papaconstantinou J 1998 Effects of age on the
posttranscriptional regulation of CCA AT/enhancer binding protein
and CCA AT/enhancer binding protein beta isoform synthesis in control
and LPS-treated livers. Mol Biol Cell 9:14791494[Abstract/Free Full Text]
-
Hattori M, Tugores A, Veloz L, Karin M, Brenner D 1990 A
simplified method for the preparation of transcrip-tionally active
liver nuclear extracts. DNA Cell Biol 10:777781
-
Dignam JD, Lebovitz RM, Roeder RG 1983 Accurate transcription
initiation by RNA polymerase II in a soluble extract from isolated
mammalian nuclei. Nucleic Acids Res 11:14751489[Abstract]
-
Song CS, Jung M-H, Kim SC, Hassan T, Roy AK, Chatterjee B 1998 Tissue-specific and androgen-repressible regulation of the rat
dehydroepiandrosterone sulfotransferase gene promoter. J Biol Chem 273:2185621866[Abstract/Free Full Text]
-
deWet JR, Wood KV, Deluca M, Helinski DR, Subramani S 1987 Firefly luciferase gene: structure and expression in mammalian cells.
Mol Cell Biol 2:10441051