Multiple Androgen Response Elements and a Myc Consensus Site in the Androgen Receptor (AR) Coding Region Are Involved in Androgen-Mediated Up-Regulation of AR Messenger RNA
Jennifer M. Grad,
Jia Le Dai1,
Shu Wu2 and
Kerry L. Burnstein
Department of Molecular and Cellular Pharmacology University of
Miami School of Medicine Miami, Florida 33101
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ABSTRACT
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The androgen receptor (AR) gene is
transcriptionally regulated by AR (autoregulation); however, the
androgen response elements (AREs) required for this process have not
been found in the AR promoter or in the 5'-flanking region. We
previously showed that the AR cDNA contains AREs involved in AR mRNA
autoregulation and that auto(up)regulation is reproduced in PC3 cells
(a human prostate cancer cell line) expressing the human AR cDNA driven
by a heterologous promoter. A 350-bp fragment of the AR cDNA contains
the requisite AREs (ARE-1 and ARE-2) and, when linked upstream of a
reporter gene, confers androgen inducibility in a cell-specific manner.
Here we report that, although an AR cDNA harboring silent mutations of
ARE-1 and ARE-2 produces a transcriptionally active AR, AR mRNA encoded
by this mutant cDNA is not up-regulated in androgen-treated PC3 cells.
Thus, ARE-1 and ARE-2 are essential for androgen-mediated up-regulation
of AR mRNA in this model. Since ARE-1 and ARE-2 are located on separate
exons (exons D and E) in the AR gene, we evaluated these AREs in their
native context, a 6.5-kb AR genomic fragment. Androgen regulated the
6.5-kb AR genomic fragment and the 350-bp region of the AR cDNA at
comparable levels, suggesting that sequences in exons D and E are
likely to be involved in androgen-mediated up-regulation of the native
AR gene. Furthermore, androgen regulated both responsive regions in
U2OS cells, a human osteoblastic cell line that exhibits
androgen-mediated up-regulation of native AR mRNA. DNAse I footprinting
of the 350-bp region with recombinant AR (DNA- and ligand-binding
domains) suggested the presence of additional AREs. Gel shift analyses
and mutational studies showed that maximal androgen regulation and AR
binding were dependent on the integrity of four AREs (ARE-1, ARE-1A,
IVSARE, and ARE-2). While the presence of multiple, nonconsensus AREs
is common among other androgen-regulated enhancers, the
androgen-responsive region of the AR gene is unique because it contains
exonic AREs. DNA binding studies with nuclear extracts were performed
to determine whether non-AR transcription factors contribute to
androgen regulation of the 350-bp region. These studies, in conjunction
with mutational analysis and reporter gene assays with dominant
negative Myc and Max expression vectors, showed that Myc and Max
interaction with a Myc consensus site is required for androgen
regulation of the 350-bp fragment. These results represent a novel
interaction between AR and the Myc family of proteins and support a
model of androgenic control of AR mRNA via AR and Myc family
interaction with a unique internal androgen-responsive region harboring
multiple exonic regulatory sequences.
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INTRODUCTION
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Androgen receptors (ARs) are ligand-activated transcription
factors that belong to the steroid/thyroid hormone receptor gene
superfamily (reviewed in Refs. 1, 2, 3). Androgen-mediated gene regulation
typically occurs through AR interaction with specific sequences within
androgen target genes termed androgen response elements (AREs). The
location, sequence, and number of AREs associated with a given androgen
target gene varies (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17), although androgen-responsive regions
typically contain multiple nonconsensus AREs (7, 15, 16, 17). AREs have
been identified in regions proximal to the target gene promoter,
several kilobases upstream from the promoter, in introns, and, in at
least two cases, exons (4, 5, 6, 7, 8, 9, 10, 11, 12). Additionally, several kilobases
separate individual AREs in some androgen-regulated genes. For example,
the androgen-regulated prostate-specific antigen (PSA) gene contains
multiple upstream AREs that are separated by approximately 4.5 kb (11, 15). Some genes contain AREs that form part of a complex regulatory
region containing binding sites for other transcription factors.
Regulatory regions of the rat 20-kDa protein gene, the rat C3(1) gene
of the prostatic binding protein, the mouse sex-limited protein
(Slp) gene, and the human prostatic-specific kallikrein gene
contain other regulatory sequences in addition to multiple AREs (7, 8, 10, 16, 17, 18, 19, 20, 21).
Androgens regulate the expression of a variety of genes including the
AR gene itself (autoregulation) (22, 23, 24). Androgen treatment of
castrated animals or treatment of AR-containing cell lines results in
tissue- and cell-specific AR mRNA autoregulation. In many tissues and
cell lines, androgen promotes down-regulation of AR mRNA; however,
androgen-mediated up-regulation occurs in several cell lines and
tissues (24, 25, 26, 27, 28). The mechanism of AR mRNA autoregulation was studied
in detail in the human prostate cancer cell line, LNCaP (29). Nuclear
run-on assays showed that down-regulation of AR mRNA is due to
decreased AR mRNA transcription (29, 30). However, studies utilizing
several cell lines failed to identify AREs in the AR gene promoter and
up to 7 kb of upstream sequences (30, 31, 32). Analysis of androgen
regulation of AR promoter activity in U2OS, an osteosarcoma cell line
that exhibits androgen-mediated AR mRNA up-regulation (3- to 4-fold),
showed only a very slight induction (<2-fold) of reporter gene
activity (24). Thus, the promoter and upstream regions of the AR gene
appear to lack AREs sufficient to account for AR mRNA
autoregulation.
We have previously shown that AR mRNA autoregulation is reproduced in
cells expressing a human AR cDNA and that this response is due to AREs
within the cDNA (4, 33, 34). Androgen-mediated down-regulation of the
AR cDNA-encoded mRNA occurs in certain transfected cell lines including
COS 1 and LNCaP (34). In contrast, androgenic up-regulation of
cDNA-encoded AR mRNA occurs in the human prostate cancer cell lines PC3
and DU145 (33). This androgen-mediated up-regulation of AR mRNA is
transcriptional and cell specific (Refs. 4, 33 and our
unpublished data). Our laboratory has focused on understanding the
molecular determinants of androgen-mediated up-regulation of AR mRNA.
We found that a 350-bp fragment of the human AR cDNA confers full
androgen-induction of a reporter gene in PC3 and DU145 cells. Two AREs,
ARE-1 and ARE-2, were identified as essential for maximal androgen
inducibility of the 350-bp region (4).
In the present study, we show that ARE-1 and ARE-2 are required for
androgen-mediated up-regulation of AR mRNA encoded by the cDNA. An AR
genomic fragment of approximately 6.5 kb containing ARE-1 and ARE-2,
like the 350-bp cDNA fragment, was androgen regulated, indicating that
these AREs function in the context of the native AR gene. In
vitro binding studies with PC3 cell nuclear extracts and an AR
fusion protein coupled with reporter gene assays showed that two
additional AREs and a Myc site are required for AR binding and
regulation of the 350-bp region. The requirements for the Myc consensus
site and functional Myc and Max proteins for maximal androgen
regulation of the 350-bp region suggests novel cross-talk between AR
and a Myc-Max heterodimer in the autoregulation of AR mRNA.
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RESULTS
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Silent Mutation of ARE-1 and ARE-2 in the AR cDNA Eliminates
Androgen Regulation of AR mRNA
To determine the role of ARE-1 and ARE-2 in androgenic
up-regulation of AR mRNA encoded by the AR cDNA, we created silent
mutations of ARE-1 and ARE-2 in the context of the entire AR cDNA.
Nucleotides were chosen for mutagenesis that would destroy each ARE
without altering the amino acid sequence of the resulting AR protein
(35). The mutant AR cDNA bearing these silent mutations was cloned into
the identical CMV promoter-driven cDNA expression vector used to
express the wild-type AR and termed CMVhARmut. CMVhARmut encodes a
functional receptor as assessed by reporter gene assays using mouse
mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT)
(Fig. 1A
). ARs encoded by the wild-type
expression vector, CMVhAR, and CMVhARmut transactivate MMTV-CAT in an
R1881 dose-responsive manner (Fig. 1A
). RNAse protection assays showed
that, unlike the wild-type AR expression vector, androgen treatment of
PC3 cells transiently expressing CMVhARmut did not result in
up-regulation of AR mRNA (Fig. 1B
). Basal levels of AR mRNA expression
were not affected by mutation of ARE-1 and ARE-2 (compare lanes CMVhAR
and CMVhARmut in the absence (-) of R1881). Thus, ARE-1 and ARE-2 are
essential for androgen-mediated up-regulation of AR mRNA in PC3 cells
expressing the AR cDNA.

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Figure 1. ARE-1 and ARE-2 Are Required for Androgen-Mediated
Up-Regulation of AR mRNA
Panel A, PC3 cells were transfected with MMTV-CAT, CMVß-gal, and
either the wild-type AR expression vector, CMVhAR, or an AR cDNA
bearing silent mutations of ARE-1 and ARE-2, CMVhARmut. Cells were
cultured in the presence or absence of R1881 for 40 h. CAT assays
were performed using cell extracts containing equivalent amounts of
ß-galactosidase activity. Panel B, PC3 cells were transfected with
CMVhAR or CMVhARmut. Twenty four hours after transfection, cells were
cultured for 20 h in the presence (+) or absence (-) of R1881.
RNase protection assays were performed to measure AR mRNA and GAPDH
mRNA levels. Radiolabeled antisense RNA specifically protected by AR
mRNA and GAPDH mRNA were 200 nucleotides and 110 nucleotides,
respectively. The experiments were performed twice and the results were
the same.
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The finding that MMTV-CAT induction is equivalent in cells expressing
the wild-type or silent mutant AR cDNA suggests that sufficient levels
of AR are produced by the transfected mutant cDNA for target gene
transactivation. Androgen-mediated up-regulation of AR protein occurred
in PC3 cells expressing the silent mutant AR (data not shown) and is
likely due to posttranscriptional stabilization of AR by ligand binding
(36).
ARE-1 and ARE-2 Are Functional in the Context of a 6.5-kb Genomic
Fragment of the AR Gene
ARE-1 and ARE-2 are located in exons D and E, respectively, and
are separated by intron 4 in the AR gene (Fig. 2
). To determine whether these exonic
regulatory elements function in their native arrangement, we cloned the
genomic fragment of the AR gene consisting of exon D, intron 4, and
exon E into the thymidine kinase (tk) promoter-driven reporter plasmid
pBLCAT2. This 6.5-kb fragment was obtained using genomic DNA as a
template and long-range PCR with primers targeting the 5'-end of exon D
and the 3'-end of exon E of the human AR gene. A single band of
approximately 6.5 kb was detected and subsequently cloned into pBLCAT2
upstream of the tk promoter (data not shown). To confirm that the
6.5-kb genomic fragment contains the appropriate region of the AR gene,
we sequenced approximately 0.7 kb on each end. The exonic sequences,
intron/exon splice sites, and the first 50 bp of each end of intron 4
were identical to those sequences reported by Lubahn et al.
(37).

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Figure 2. Location of ARE-1 and ARE-2 in the Human AR Gene
The hatched rectangles and straight lines
represent AR gene exons and introns, respectively. ARE-1 and ARE-2 are
noted with asterisks. The 6.5-kb genomic
androgen-responsive region contains exon D, intron 4, and exon E. This
6.5-kb fragment was inserted upstream of the tk promoter in the
reporter plasmid pBLCAT2, resulting in I4-CAT.
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The reporter plasmid containing this 6.5-kb AR genomic fragment
(I4-CAT) is androgen regulated in PC3 and an osteosarcoma cell line
U2OS (Fig. 3
), but not in the human
prostate cancer cell line LNCaP (data not shown). Regulation of I4-CAT
is comparable to that of the reporter plasmid containing the 350-bp
cDNA fragment, 350WT-CAT (
8 fold; P > 0.05),
suggesting that ARE-1 and ARE-2 can function in their genomic
arrangement. Furthermore, I4-CAT exhibits the same cell specificity as
350WT-CAT (Fig. 3
and data not shown). This cell specificity is
significant because, as we have shown, androgen treatment results in
the up-regulation of AR mRNA encoded by a wild- type AR cDNA in PC3
cells (Fig. 1B
and Ref. 33) and up-regulation of native AR mRNA in U2OS
cells (Ref. 24 and J. Grad and K. L. Burnstein, unpublished data).
In contrast, both the native (23, 29, 30) and cDNA-encoded AR mRNAs are
down-regulated in LNCaP cells (34). Therefore, these findings are
consistent with a role for the exonic elements ARE-1 and ARE-2 in
cell-specific, androgen-mediated up-regulation of native AR mRNA.

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Figure 3. Androgen Regulation of the 6.5-kb AR Genomic and
350-bp AR cDNA Fragments Containing ARE-1 and ARE-2
PC3 and U2OS cells were cotransfected with CMVhAR, CMVß-gal, and
350WT-CAT or I4-CAT and cultured for 40 h in the absence (-) or
presence (+) of R1881 (50 nM). 350WT-CAT contains the
350-bp AR cDNA fragment, and I4-CAT contains the 6.5-kb genomic AR
fragment, both ligated upstream of the tk promoter in the parent vector
pBLCAT2. CAT assays were performed using cell extracts containing
equivalent amounts of ß-galactosidase activities. Representative
experiments (three for PC3 and five for U2OS) are shown.
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DNAse I Footprinting of the 350-bp Androgen-Responsive Region
Reveals an Additional AR Binding Site between ARE-1 and ARE-2
We previously demonstrated by gel shift assay that an AR fusion
protein consisting of the human AR DNA and steroid-binding domains
fused to glutathione-S-transferase (GST-AR) expressed in
E. coli binds specifically to the 350-bp fragment (4). To
map potential sequences within the 350-bp region involved in contacting
AR, we performed DNAse I footprinting studies using the amino
terminal-deleted AR, produced by thrombin cleavage of GST-AR
(Fig. 4
). These experiments revealed the
presence of three AR-binding sites encompassing ARE-1, ARE-2, and a
region between these AREs termed intervening sequence (IVS) (Fig. 5A
and 5B
, lanes labeled "350WT"). AR
binding to these regions was examined by using 350-bp probes labeled at
the upstream HindIII site (to visualize ARE-1 and IVS, Fig. 5A
) and at the downstream XbaI site (to visualize ARE-2,
IVS, and ARE-1, Fig. 5B
). The first pair of lanes in each panel shows
the footprints obtained with a wild-type 350-bp probe (350WT). To
assess the contribution of ARE-1 and ARE-2 to AR binding, we analyzed
DNAse I footprinting of 350-bp fragments harboring mutations at either
ARE-1 (mARE-1) or ARE-2 (mARE-2). Mutation of ARE-1 (lanes marked
mARE-1) resulted in no footprint at the region encompassing ARE-1 and
substantially decreased (5090%) AR binding to both ARE-2 and IVS
(Fig. 5
, A and B; hatched lines mark the corresponding
regions protected on 350WT). Similarly, mutation of ARE-2 (mARE-2)
resulted in loss of the footprint at the region encompassing ARE-2 and
decreased (5075%) footprinting at ARE-1 and IVS (Fig. 5
, A and B;
hatched lines mark the corresponding regions protected on
350WT probe). The same results were obtained when a GST-AR
fusion protein was used in footprinting analysis of wild-type and
mutated 350-bp fragments (data not shown). The end-labeled IVS region
alone is not bound by GST-AR (Fig. 5C
; hatched lines show
the corresponding region protected on the wild-type fragment)
suggesting that AR binding to the IVS region requires the presence of
ARE-1 and ARE-2. The results of the DNAse I footprinting experiments
are summarized in Fig. 6
.

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Figure 4. Isolation of an Amino Terminal-Deleted AR by
Thrombin Cleavage of GST-AR
Coomassie-stained gel of GST-AR (DNA- and ligand-binding domains)
fusion protein, and amino terminal-deleted AR produced by thrombin
cleavage is shown. GST-AR bound to glutathione Sepharose 4B was
incubated in the absence (-) or presence (+) of thrombin as described
in Materials and Methods. AR protein was recovered in
the flowthrough. Size markers are indicated (M). GST-AR is a protein of
approximately 78 kDa and the amino terminal-deleted AR is about 52 kDa.
The protein of about 30 kDa is probably GST. A contaminating
Escherichia coli protein (non-spec) of approximately 64
kDa was frequently present. Preparations of GST-AR chosen for use
without further purification contained a higher amount of GST-AR
relative to contaminating proteins.
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Figure 5. Mutation of Individual AREs Decreases AR Binding at
All Other AREs
DNAse I footprinting studies were performed with the wild-type 350-bp
region (350WT), 350-bp fragments with mutations at ARE-1 (mARE-1) or
ARE-2 (mARE-2), and the intervening region of the AR cDNA between ARE-1
and ARE-2 (IVS). The designated fragments (350WT, mARE-1, mARE-2) were
end labeled, incubated with BSA or an amino terminal-deleted AR
(consisting of the DNA-binding domain and ligand-binding domains) and
subjected to in vitro DNAse I footprinting. The IVS
fragment was incubated with GST-AR or GST. Protected regions
(footprints) detected using the 350WT probe are designated by
solid vertical lines. When mutant probes (mARE-1,
mARE-2) were used, the regions corresponding to footprints on 350WT are
designated by hatched lines. Data were quantified by
phosphorimage analysis of dried gels. The amount of radioactivity in
each protected region in the AR lanes was compared with the analogous
region in the BSA or GST control lane and normalized to a region
outside the footprint. A, The 350-bp fragments (350WT, mARE-1, and
mARE-2) were end labeled at artificially generated
5'-HindIII sites to view footprints over ARE-1 and IVS.
B, The 350-bp fragments (350WT, mARE-1, and mARE-2) were end labeled at
the 3'-XbaI site to view footprints over ARE-2 and IVS.
C, No binding of GST-AR to the IVS was observed.
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Figure 6. Summary of AR Footprinting of the 350-bp AR cDNA
Fragment
DNA sequences protected from DNAse I digestion are shown in
boldface and AREs are double underlined.Putative AREs are single underlined. The
footprint located between ARE-1 and ARE-2 is designated IVS. Numbering
is based on the human AR cDNA (58 ) with +1 located 42 bp 5' to the
translation start site.
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DNAse I footprinting results suggest the presence of AREs in addition
to ARE-1 and ARE-2. The IVS binding site contains a sequence with
similarity to the consensus ARE
[(5-GAGACAgctTGTACA-3')
in which the consensus nucleotides (35) are underlined]. The
consensus ARE is a bipartite, partially palindromic element with a
three-nucleotide spacer. However, binding of AR to the IVS region
requires ARE-1 and ARE-2, as the IVS alone is not protected by GST-AR
(Fig. 5C
). Furthermore, as shown in Fig. 1B
, mutations of ARE-1 and
ARE-2 in the AR cDNA are sufficient to abrogate androgen regulation of
AR mRNA encoded by the mutated cDNA, suggesting that an ARE in the IVS
cannot function independently. The DNAse I footprinting experiments
described above showed that the ARE-1 footprint is approximately 57 bp,
which is larger than other reported AR binding sites (e.g.
Refs. 12, 13). Thus, an additional ARE may be located near ARE-1 and
contribute to the large protected region. A 15-bp sequence with
homology to the ARE consensus is located within the ARE-1 footprint,
[(5-GGTGTAgtgTGTGCT-3');
consensus nucleotides are underlined (35)]. Although neither of these
putative AREs possess the well conserved "right half" sequence,
TGTYCT, found in the majority of AREs, the two elements are fairly
similar to the consensus bipartite ARE (8 of 12 nucleotides) (35).
Two Additional AREs Contribute to AR Binding and Androgen
Regulation of the 350-bp Androgen-Responsive Region
To determine the contributions of these putative AREs to androgen
regulation of the 350-bp AR cDNA fragment (350WT-CAT), we introduced
mutations in each putative response element in the 350-bp region. A
mutation was also made outside the footprinted areas as a mutagenesis
control (mc350). These 350-bp fragments, mARE-1A (mutation in the
putative ARE near ARE-1), mIVSARE (mutation in the element within the
IVS), and mc350, were inserted upstream of the tk promoter in the
reporter plasmid pBLCAT2. The resulting reporter plasmids, mARE1A-CAT
and mIVSARE-CAT, displayed a substantial reduction of androgen
inducibility (80% and 87% decrease, respectively) compared with the
350WT-CAT and mc350-CAT (Fig. 7A
).
These data suggest that both ARE-1A and IVSARE are bona fide AREs and
are required for maximal androgen regulation of the 350-bp region.

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Figure 7. Two Additional AREs (ARE-1A and IVSARE) Are
Required for Maximal Androgen Regulation and AR Binding of the 350-bp
Androgen-Responsive Region
Panel A, PC3 cells were cotransfected with CMVhAR, the indicated CAT
construct containing the 350-bp androgen-responsive region (350WT),
ARE-mutant (mIVSARE, mARE-1A) or mutagenesis control (mc350) and
CMVß-gal. Cells were cultured in the presence (+) or absence (-) of
R1881 (50 nM) for 40 h. CAT assays were performed
using cell extracts containing equivalent amounts of ß-galactosidase
activities. Representative autoradiograms are shown. Fold induction is
the ratio of CAT activity observed in the presence and absence of R1881
treatment. Each bar represents the mean ± SEM of
three to four experiments. Panel B, Gel shift assays were performed as
described in Materials and Methods using a purified
GST-AR fusion protein (500 ng) and radiolabeled 350WT fragment in
the presence of increasing amounts (0-, 50-, 100-, or 250-fold molar
excess) of unlabeled wild-type or mutant 350-bp fragments. Purified GST
is included as a control. The free probe and AR-DNA complexes are
indicated in a representative autoradiogram. Radioactivity in
DNA-protein complexes was quantified by phosphorimage analysis and the
percent of GST-AR binding (binding in presence of competitor/binding in
the absence of competitor) plotted in the lower portion of panel
B. A comparably sized fragment of the AR cDNA that does not
contain AREs served as a nonspecific competitor. In addition, a 350-bp
fragment harboring a mutation in sequences adjacent to the IVSARE
footprint (mutagenesis control, mc350) was tested. Each point
represents the mean ± SEM of three independent
determinations for each concentration of the indicated competitor,
except for the nonspecific and mc350 competitors, which were performed
once.
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The effects of mutating these AREs (ARE-1A and IVSARE) on AR
binding were evaluated by gel shift assay using radiolabeled 350WT and
increasing amounts of unlabeled mutated 350-bp fragments as
competitors. The 350-bp fragments containing mutations in ARE-1A or
IVSARE were less effective competitors of AR-350 bp complex formation
than the wild-type fragment (Fig. 7B
). A nonspecific competitor (a
comparably sized portion of the AR cDNA that lacks AREs) acts as a very
poor competitor (Fig. 7B
). Although both of the mutated 350-bp
fragments were weaker competitors than 350WT, there were subtle
differences between the ability of each mutant fragment to compete for
AR binding (e.g. mARE-1A vs. mIVSARE), suggesting
that the AREs are not bound equally by the recombinant AR. Although
these studies do not provide quantitation of the affinity of AR binding
to each ARE, they do illustrate that mutation of either ARE decreases
DNA-AR interaction. Together, these studies and our previously
published results (4) demonstrate that ARE-1, ARE-2, ARE1-A, and IVSARE
each contribute to maximal AR protein binding and androgen regulation
of the 350-bp region.
A Consensus Site for Myc Is Required for Maximal AR Binding and
Androgen Regulation of the 350-bp Region
Since auxiliary transcription factors contribute to the regulation
of many steroid target genes, we performed DNAse I footprinting
experiments with nuclear extracts from PC3 cells to determine whether
non-AR transcription factors interact with the 350-bp region. These
studies revealed subtle protection of a region that contains a perfect
consensus site for a variety of transcription factors (CACGTG; E box),
including the protooncogene Myc and the related proteins Max, Mad, and
Mxi (reviewed in Refs. 38, 39) (Fig. 8A
). The E box
overlaps with the IVSARE by 2 bp. To determine whether PC3 nuclear
protein complexes bind specifically to this Myc consensus sequence, we
performed additional DNAse I footprinting experiments with a 350-bp
fragment in which the Myc consensus site was mutated. No footprint was
observed over the mutated Myc site (Fig. 8B
). To confirm the
specificity of the DNA-nuclear protein interaction, gel shift analyses
were performed using 350WT as a probe and unlabeled
oligonucleotides containing a consensus Myc site or mutated Myc site
(CAGCTG; bolded nucleotides were mutated) as
competitors. These studies identified two DNA-protein complexes that
were specifically competed by the Myc consensus site oligonucleotide,
but not by the mutated oligonucleotide (Fig. 8C
). Thus, proteins
present in PC3 cell nuclear extracts specifically recognized the Myc
consensus sequences in the 350-bp region. To determine the
contributions of the Myc consensus site to androgen regulation of the
350-bp region and AR binding to this fragment, the site was mutated in
the context of the 350-bp fragment without disrupting the IVSARE. The
resulting plasmid, mMyc-CAT, displayed decreased androgen inducibility
(89% decrease) compared with 350WT-CAT in PC3 cells
(Fig. 9A
). Similar to fragments containing a mutated
ARE, the mMyc 350-bp fragment was also a less effective competitor than
350WT for binding to GST-AR in gel shift assays (Fig. 9B
). It is
important to note that all four core ARE sequences are intact in the
mMyc-CAT construct. Similarly, the minimal Myc sequences are intact in
the mIVSARE-CAT construct.

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Figure 8. DNAse I Footprinting and Gel Shift Analysis Reveal
the Presence of a Consensus Binding Site for the Myc Family of
Protooncogenes within the 350-bp Androgen-Responsive Region
A, DNAse I footprinting of the 350-bp fragment (end-labeled at the
artificially generated 5'-HindIII site) was performed
using nuclear extracts from PC3 cells (20 µg). Nuclear extract buffer
control (equal amount of heat-inactivated nuclear extract), GST, and
GST-AR lanes are shown for comparison. M13 primers were included as
size markers. The thin vertical line in the GST-AR lane
indicates the IVSARE footprint. The thick bar in the PC3
nuclear extract lane (PC3 NE) marks the region protected from DNAse
digestion, which contains a consensus site for the Myc family of
protooncogenes. The Myc consensus sequences are
underlined, and regions that overlap with the
GST-AR-protected IVSARE are boldface. B, DNAse I
footprinting of the 350WT fragment and a 350-bp region with a mutated
Myc site (mMyc) (end-labeled at the artificially generated
3'-XbaI site) was performed using nuclear extracts from
PC3 cells (10 µg and 20 µg). M13 primers were used as size markers
but are not shown. Nuclear extract buffer control (20 µg of
heat-inactivated nuclear extract) is shown for comparison. The region
indicated by a bracket corresponds to the same protected
region observed in panel A. C, Gel shift assays were performed as
described in Materials and Methods using nuclear
extracts from PC3 cells (5 µg) and radiolabeled 350WT fragment in the
presence or absence of 25- and 50-fold molar excess of the indicated
unlabeled oligonucleotide competitors [Myc binding site oligo (WT) or
a mutated Myc binding site oligo (mutant)]. Protein-DNA complexes that
were specifically competed away are indicated by arrows.
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Figure 9. A Myc Consensus Site Is Required for Maximal
Androgen Regulation and Androgen Binding of the 350-bp
Androgen-Responsive Region
A, PC3 cells were cotransfected with CMVhAR, the indicated CAT
construct [350WT (wild type) or mMyc (mutated Myc)] and CMVß-gal
and treated for 40 h with 50 nM R1881. CAT assays were
performed using samples containing equivalent amounts of
ß-galactosidase activities. Fold induction is the ratio of CAT
activity observed in the presence and absence of R1881 treatment (50
nM). Each bar represents the mean ±
SEM of three to four experiments. B, Gel shift assays using
purified GST-AR fusion proteins in the presence of increasing amounts
of unlabeled wild-type or mutant Myc fragments were performed as
described in Materials and Methods. The gels were
quantitated by phosphorimage analysis, and the percent of total binding
(GST-AR binding in presence of competitor/binding in absence of
competitor) was determined. Nonspecific DNA competitor and the mc350-bp
fragment (mutagenesis control) are included. Each point represents the
mean ± SEM of three experiments except for
experiments testing the nonspecific and mutagenesis control (mc)
fragments, which were conducted once and shown in Fig. 7B .
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To determine whether a functional Myc/Max heterodimer contributes to
androgen regulation of the 350-bp androgen-responsive region, we used
expression vectors encoding dominant negative Myc and dominant negative
Max (Ref. 40 ; generously provided by Dr. B. Amati). A transcriptionally
active Myc complex is a heterodimer comprised of Myc and its binding
partner Max. Myc and Max proteins interact via a basic helix-loop-helix
leucine zipper (HLH-LZ) domain. Dominant negative mutants of Myc
(MycRx) and Max (MaxRx) were created by reciprocal exchange of the
HLH-LZ domains of Myc and Max (40). Thus, the MycRx mutant contains
the Max HLH-LZ domain and binds tightly to wild-type Myc. The resulting
MycRx-Myc complex does not bind DNA and is inactive (40). MycRx thereby
prevents wild-type Myc from forming functional complexes with wild-type
Max. The MaxRx mutant contains a Myc HLH-LZ domain and forms
nonfunctional complexes with wild-type Max. The presence of MaxRx
results in the titration of wild-type Max via the formation of stable
but transcriptionally inactive MaxRx-Max heterodimers (40). When MycRx
and MaxRx mutants are introduced together, MycRx-MaxRx heterodimers can
be formed and these heterodimers are functional (40).
We found that introduction of either MycRx or MaxRx (1.0 µg per 60-mm
dish) abrogates androgen regulation of reporter plasmids containing
either the 350-bp cDNA or 6.5-kb genomic fragment-containing reporter
plasmids (Fig. 10
, A and B). The same results were
obtained when 0.5 µg of dominant negative plasmids was used (data not
shown). Androgen regulation was restored when MycRx and MaxRx were
transfected together. These data suggest that functional Myc and Max
are required for androgen regulation of these responsive regions. To
test the possibility that dominant negative Myc or Max influences the
regulation of other androgen-responsive regions, we examined androgen
regulation of MMTV-CAT in cells cotransfected with wild-type Myc and
Max, as well as the dominant negative expression vectors, MycRx and
MaxRx. None of these vectors had any effect on androgen-mediated
transactivation of MMTV-CAT (Fig. 10C
). Androgen regulation of a
3xHRE-CAT construct was also not affected by introduction of these
expression vectors (data not shown). These studies suggest that Myc and
Max (possibly as a heterodimer) are involved in androgen regulation of
the 350-bp and 6.5-kb regions.

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Figure 10. Dominant Negative Myc or Max Blocks Androgen
Regulation of the Androgen-Responsive Regions
Panel A, PC3 cells were cotransfected with CMVhAR, 350WT-CAT,
CMVß-gal, and 1.0 µg of the indicated plasmid [parent vector
(pBJ3), dominant negative Myc (MycRx), or dominant negative Max
(MaxRx)]. Cells were treated for 40 h with 50 nM
R1881. CAT assays were performed using samples containing equivalent
amounts of ß-galactosidase activities. Fold induction is defined as
the ratio of CAT activity observed in the presence and absence of R1881
treatment. Each bar represents the mean ± SEM of
three to four experiments. Panel B, The experiment was conducted as
described in panel A except I4-CAT was used in place of 350WT-CAT. A
histogram of data from three independent experiments is shown. Panel C,
The experiment was conducted as described in panel A except MMTV-CAT
was tested in place of 350WT-CAT. A histogram of data from two
independent experiments is shown.
|
|
To determine whether Myc and Max bind to the 350-bp region, we tested
the ability of anti-Max and anti-Myc antibodies to alter the mobility
of protein-DNA complexes in gel shift experiments (supershift).
Although inclusion of anti-Max antibodies did result in a supershifted
band, we failed to detect any up-shifted bands using several different
anti-Myc antibodies (data not shown). Since it is possible that the
available anti-Myc antibodies recognize denatured Myc protein but not
the native form, we used gel shift assays coupled with Western blotting
to determine whether Myc and Max bind to the 350-bp region. For these
studies, we conducted gel shift assays using PC3 cell nuclear extracts
and radiolabeled 350WT fragment. The DNA-protein complexes and the
indicated regions of the gel were excised and subjected to SDS-PAGE and
immunoblotting. Anti-Max antibodies specifically detected a protein
with a similar molecular mass as Max (
22-kDa); and anti-Myc
antibodies specifically detected a protein with a similar size to Myc
(
62 kDa) (Fig. 11
). Although the gel was UV
cross-linked, only free Myc or Max protein was detected, as any
Myc/Max-DNA complexes would have migrated more slowly. These
experiments show that Myc and Max bind to the 350-bp region.

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Figure 11. Myc and Max Bind to the 350-bp DNA Fragment
Preparative gel shifts (100 µg of PC3 nuclear extracts) were
conducted as described in Materials and Methods. Note
that the gels were run longer to further separate and distinguish the
nuclear protein-DNA complexes (duplicate preparative gels were run; a
representative autoradiogram is shown). The 5%
nondenaturing gels were exposed to x-ray film, and DNA-protein
complexes were visualized by autoradiography of the wet gel (not
shown). The bands corresponding to those specifically competed by Myc
oligonucleotides (Fig. 8C ) as well as the indicated additional bands
were excised and subjected to SDS-PAGE, transferred to nitrocellulose,
and probed with anti-Max or anti-Myc antibodies. A single 22-kDa band
(Max) was detected by the anti-Max antibody; the anti-Myc antibody
detected a 62-kDa band (Myc).
|
|
 |
DISCUSSION
|
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The overall goal of this work is to identify the relevant
regulatory sequences involved in androgen-mediated up-regulation of AR
mRNA. Since AREs have not been identified in the AR gene promoter and
upstream flanking region (30, 31, 32), and because autoregulatory elements
for both glucocorticoid and estrogen receptors are present in the
cognate receptor gene-coding region (41, 42), we investigated the role
of AR gene exonic sequences in the up-regulation of AR mRNA induced by
androgen. We previously reported that the AR cDNA contains AREs and is
sufficient for the cell-specific up-regulation of AR mRNA (4, 33). Here
we focused on identifying the specific exonic sequences, both AREs and
non-AR transcription factor-binding sites, that contribute to
androgen-mediated up-regulation of AR mRNA. We identified four AREs and
a Myc consensus site in the AR coding region that are involved in AR
binding and androgen-mediated up-regulation of AR mRNA encoded by the
AR cDNA. It is of interest that these AREs and Myc site appear to be
well conserved (0 or 1 mismatches) among a number of species from
rodents and monkeys to birds and frogs (data not shown). While it is
difficult to interpret the significance of this conservation given the
exonic location of these sequences, conservation of these sequences is
consistent with a role for these regulatory elements in autoregulation
of AR mRNA.
One caveat of the AR binding studies with the 350-bp AR cDNA fragment
is that the AREs are not in their native arrangement, where three AREs
are present on exon D and one ARE is present on exon E. Thus, the
effects of mutating one ARE on AR binding to adjacent AREs may not
extend to the native gene. However, since the AR genomic fragment
encompassing these elements confers androgen inducibility to a reporter
gene, the exonic elements are functional when separated by intron 4.
Furthermore, the 6.5-kb genomic and 350-bp cDNA androgen-responsive
regions show the same cell specificity: both regions are androgen
regulated in U2OS and PC3 cells (Fig. 3
), but not in LNCaP cells (data
not shown). These findings are significant because U2OS and PC3 cells
exhibit androgen-mediated up-regulation of AR mRNA (24, 33), whereas
LNCaP cells exhibit androgen-mediated down-regulation of AR mRNA (23, 29, 30). Thus, these androgen-responsive regions maintain the cell
specificity that is predicted for elements involved in the
up-regulation of the native AR gene.
The androgen-responsive region of the AR gene contains multiple,
nonconsensus AREs located in two exons separated by an intron of
approximately 6.2 kb. Multiple, dispersed steroid-response elements
appear to be a characteristic of many steroid-regulated genes. In
several steroid hormone regulated genes, response elements are located
several kilobases from each other and from the target gene promoter.
For example, estrogen regulation of the rat progesterone receptor (PR)
gene occurs via multiple, synergistic estrogen response elements (EREs)
dispersed over more than 4 kb of the rat PR gene (43). Several of these
EREs are not estrogen regulated individually, but require the presence
of the other EREs (43). Maximal androgen regulation of the PSA gene
requires two distinct ARE-containing regions, one proximal to the
promoter and one about 4.2 kb upstream from the proximal regulatory
region (11, 15, 44). Thus, our finding that multiple AREs contained in
an intragenic 6.5-kb fragment confer androgen regulation is consistent
with some features of other steroid-regulated genes. Androgen
regulation of target gene expression typically involves cooperativity
among multiple nonconsensus AREs (7, 14, 15, 16, 17, 44). The finding reported
here that mutation of any ARE or of the Myc consensus site results in a
drastic reduction of androgen regulation of the 350-bp region is
consistent with such cooperativity.
The internal regulatory region we identified in the human AR gene is
located a great distance from the AR promoter. This arrangement of
regulatory sequences is not unprecedented, as a variety of
transcription factors (including steroid receptors) act via regulatory
sequences that are downstream from the transcription start site and
separated from gene promoters by many kilobases (reviewed in Ref. 45).
For example, the
-globin gene contains an internal regulatory region
(encompassing exons 1 and 2 and intron 1) that cooperates with upstream
5'-flanking sequences to confer tissue-specific gene expression (46).
Studies in transgenic mice revealed that androgen regulation of the
ß-glucuronidase gene involves regulatory sequences present within a
large genomic fragment (encompassing intron 3 through intron 9) located
approximately 6.4 kb downstream from the ß-glucuronidase gene
promoter (5, 6). Androgen-responsive regions have also been described
within intron 1 of the rat 20-kDa protein and intron 1 of the
prostatein C3 subunit gene (7, 8, 9).
Despite the demonstration of internal cis-acting sequences
in a variety of steroid target genes, no clear mechanism for how these
sequences function has emerged. Downstream regulatory elements might
modulate transcription via a variety of mechanisms including
influencing the rate of transcription initiation or elongation. DNA
looping and characteristics of chromatin structure may facilitate
interactions between transactivators binding downstream and in the
promoter region (45). Alternatively, transactivators binding to
downstream regulatory elements may interact with an upstream enhancer
element to influence the rate of initiation. For instance, AR binding
intragenically might interact with an upstream enhancer to increase
transcription initiation from the AR gene promoter. Another possible
mechanism involves augmentation of transcriptional elongation. In this
model an enhancer protein binds transiently to a downstream element and
opens up the DNA strand, allowing more efficient elongation. Since the
androgen-responsive region of the AR gene can function upstream of a
heterologous promoter (350WT-CAT, I4-CAT), enhanced elongation is less
likely. Bridging or adaptor proteins (coregulators) are also likely to
play a role in mediating the activities of distal and proximal
regulatory elements, be they intragenic or in the 5'-flanking region
(47, 48).
Nonreceptor transcription factors can play a critical role in
steroid-mediated gene regulation; however, the mechanism by which
nonreceptor factors influence steroid receptor action is not fully
understood (16, 17, 18, 19, 49, 50). These auxiliary factors may confer
cell-specific effects through distinct combinations of factors acting
on complex regulatory regions. The tissue-specific effects of androgens
might be achieved via interactions between AR and nonreceptor
DNA-binding proteins (16, 17, 18, 19, 49, 50). We describe here a novel link
between the Myc family of transcription factors and AR in AR mRNA
autoregulation. The members of this family of proteins (Myc, Max, Mad
14, Mxi, etc.) regulate gene expression from CACGTG elements through
a complex array of homo- or heterodimers (reviewed in Refs. 38, 39).
Members of the Myc family are deregulated in a variety of human
malignancies (38, 39); and a role for this family of proteins is well
established in proliferation, differentiation, oncogene transformation,
and programmed cell death (38, 40, 51). Differential AR mRNA
autoregulation occurring during development (52), for example, may be
related to changes in the relative levels of Myc and related family
members. AR/Myc interactions may underlie androgen-mediated regulation
of AR mRNA in the developing prostate (52) where changes in functional
Myc/Max heterodimers might augment or attenuate sensitivity to
androgen. Overexpression of c-myc in prostate cancer cells
(53) may influence AR levels and androgen-stimulated proliferation.
Finally, a connection between androgen responsiveness (via
up-regulation of AR mRNA) and Myc function is intriguing as it offers
another link between AR and the cell cycle (54, 55, 56, 57).
 |
MATERIALS AND METHODS
|
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Cell Culture, Transfection, and CAT Assays
PC3 and U2OS cells (American Type Culture Collection, Manassas, VA) were routinely cultured in RPMI-1640
media or McCoys media, respectively, supplemented with 100 IU/ml
penicillin, 100 µg/ml streptomycin, 2 mM
L-glutamine (all from Life Technologies, Inc.,
Gaithersburg, MD), and, unless otherwise noted, 10% heat-inactivated
FBS (HyClone Laboratories, Inc. Logan, UT).
Cells were transfected using the calcium phosphate coprecipitation
method (4). Supercoiled plasmid DNA used for transfection was purified
by CsCl2-ethidium bromide gradient centrifugation or with
Qiagen columns (Valencia, CA). All cells were
cultured in 60-mm plates and received 500 ng CMVhAR, 10 µg CAT
reporter plasmid, and 1.0 µg ß-galactosidase expression plasmid
(CMVßgal) (to normalize for transfection efficiency). For the studies
using dominant negative Myc and Max, each 60-mm dish of cells also
received 0.5 or 1.0 µg of the parent vector pBJ3, MaxRx, or MycRx
expression vectors. Transfected cells were cultured in RPMI-1640 or
McCoys media supplemented with 2% charcoal-stripped FBS.
Charcoal-stripped FBS was prepared by incubating serum with
dextran-coated charcoal to deplete endogenous steroids.
Methyltrienolone (R1881, from DuPont NEN, Boston, MA) or
ethanol vehicle was added to the cultures either immediately after
transfection (for reporter gene assays) or 24 h after transfection
(for AR mRNA studies). Forty hours after R1881 treatment, transfected
cells were harvested in 1x reporter lysis buffer (Promega Corp., Madison, WI). ß-Galactosidase and CAT activities from
cell extracts were measured as previously described (4). After the
ß-galactosidase assays, cell extracts were heated at 65 C for 10 min.
Aliquots of cell extracts containing equivalent ß-galactosidase
activities were incubated with 1.0 mM acetyl-coenzyme A
(Roche Molecular Biochemicals, Indianapolis, IN) and 5.87
µM [14C]-chloramphenicol (DuPont NEN, specific activity, 50.0 mCi/mmol) in a total volume of 150
µl for 26 h at 37 C. Acetyl-coenzyme A (5 µl; 8 mM)
was added to each reaction after every 2 h of incubation. The
percent conversion of chloramphenicol to acetylated forms was
quantified by phosphorimage analysis using ImageQuant software
(Molecular Dynamics, Inc., Sunnyvale, CA). Statistical
analysis was performed using Students t test.
Reporter Plasmid Construction
The reporter plasmid 350WT-CAT was previously described (4). All
350-bp fragments were inserted in the 5'- to-3' orientation upstream of
the tk promoter of pBLCAT2. The numbering system used for the reporter
plasmids is based on the human AR cDNA (58) with +1 located 42 bp 5' to
the translation start site.
The I4-CAT reporter plasmid was created using an exonuclease III
digestion strategy (59). The AR genomic fragment was amplified by PCR
using human genomic DNA (Boehringer Mannheim, Indianapolis, IN) as
template and primers targeted to the 5'- and 3'-ends of exon D and E of
the AR gene, respectively. The forward primer was
5'-TGCCTGCAGGTCGACAACCCAGAAGCTGACAGTGTC-3'; the reverse primer was
5'-TCCTCTAGAGTCGACCGGTACTCATTGAAAACCAGATC-3'. After gel purification,
the 6.5-kb PCR product (50 ng) and SalI-digested pBLCAT2
were incubated with Exonuclease III (Stratagene, La Jolla,
CA) for 45 sec. The total reaction volume was raised to 50 µl with
Tris-EDTA (TE) buffer (pH 8.0), and the ligated DNA was
phenol/chloroform extracted and ethanol precipitated. The pellet was
dissolved in TE buffer (pH 8.0) and incubated at 37 C for 10 min. A
5-µl aliquot was used for transformation of Epicurian Coli XL-10 Gold
Ultracompetent cells (Stratagene).
The expression vectors, Myc, Max, MycRx, and MaxRx, and the parent
vector pBJ3 were generously provided by Dr. B. Amati (40).
RNAse Protection Assays
RNAse protection assays were performed essentially as
described previously (4) using a human AR antisense RNA probe
synthesized from a 0.7-kb HindIII-EcoRI fragment
of human AR cDNA subcloned into BluescriptIIKS
(Stratagene). The probe template was linearized with
ScaI. [
-32P]UTP (DuPont NEN, specific
activity 800 Ci/mmol)-labeled antisense RNA probes were generated by
in vitro transcription with T7 RNA polymerase using the
Maxiscript kit (Ambion, Inc. Austin, TX). One microgram of
total RNA was hybridized with both AR and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) antisense RNA probes simultaneously. The RNA
samples were treated with 0.5 U RNAse A/20 U RNAse T1 for 30 min at 37
C. RNA was precipitated and fractionated by electrophoresis through 5%
polyacrylamide gels containing 4 M urea. Probe fragments
specifically protected by AR and GAPDH mRNA are 200 and 110
nucleotides, respectively.
Mutagenesis and PCR
The expression vector CMVhARmut containing silent mutations of
ARE-1 and ARE-2 was generated by sequential PCR using CMVhAR as the
template. To create the ARE-1 silent mutation, the forward primer F9
(5'-TAGCCCCCTACGG-CTACACT-3') and backward primer B11
(5'-CGATCGAGTTCCTTGATGTAGT-3') were paired, respectively, with a
backward primer B9
(5'-GGCTCAATGGCTTCCAAGACGTTCAGAAAGATGG-3') and a
forward primer F7
(5'-CCA-TCTTTCTGAACGTCTTGGAAGCCATTGAGCC-3')
that each contain two mutated nucleotides (in boldface) to
replace the wild-type ARE-1. The reactions resulted in two PCR products
that contained overlapping sequences. These two resultant PCR products
were then amplified as templates using F9 and B11 to generate a 1.1-kb
fragment (+1367 to 2557) harboring an ARE-1 mutant. Similarly, to
generate silent mutation of ARE-2, F9 and a backward primer B10
(5'-GAGCCCCATCCAGGAATATTGAATGACAGCC-3'), and a
forward primer F8
(5'-GGCTGTCATTCAATATTCCTGGATGGGGCTC-3') and B11
were paired, respectively, using the ARE-1 mutant fragment as a
template. The resulting 1.1-kb PCR product was digested with
AocI and SfuI, and this fragment was used to
replace the corresponding wild-type fragment of the AR cDNA in the
Bluescript II vector (BSAR3.1). The AR cDNA bearing mutations in ARE-1
and ARE-2 was subcloned into the CMV2 expression vector.
The mutant reporter constructs, mARE-1A-CAT, mIVSARE-CAT, mMyc-CAT, and
mc350-CAT, were generated using the CloneAmp System (Life Technologies, Inc.). The 5'-half of each ARE was mutated
(boldface nucleotides) as follows: ARE-1A was mutated from
TGTGCT to GAATTC (mARE-1A); IVSARE was mutated from TGTACA
to GAACCA (mIVSARE). The mMyc plasmid contains a 350-bp
region mutated at the Myc site CACGTG to CAGCTG. In the
mutagenesis control plasmid (mc350-CAT), sequences were mutated from
GGGCCA to CAGCTG (14 bp 3' to the IVSARE). The
mMyc fragment used for footprinting studies (primers are noted as
MYCFOOT below) is mutated from TGTACACGTG to GAATTCCGTG. To
create the mutant constructs, a first round of PCR reactions was
performed to generate the 5'- and 3'-halves of each mutated 350-bp
region, respectively (underlined nucleotides were
mutated). The primers for the first round of PCR were the forward (F)
primer F-H3CAU (5'-CAUCAUCAUCAUGCCAGTGCCAAG-CTTAAC-3') paired with
the indicated reverse (R) primer: R-ARE1-A
(5'-GTGTCCGAATTCCACTACACCTGG CTCA-ATG-3'), R-IVSARE
(5'-CCACGTGGTTCAGCTGTCTCTCTCCCAG-3'), R-MYC
(CAGTACCAGCTGT ACAAGCTGT-CTCTCTCCCAG-3'), R-MC350
(5'-CCTTGGCAGCTGTGA-CCACGTGTAC-3'), R-MYCFOOT
(5'-CAGTACGAATTCA-GCTGTCTCTGTCCCAGTTCATT-3') to produce
the 5'-fragments of each mutant. The 3'-fragments were generated using
the reverse primer R-XbaCUA 5'-CUACUACUACUAGGATCCTCTAGACGGTAC-3')
paired with the indicated forward primer (underlined
nucleotides are mutated): F-ARE1-A
(5'-GGTGTAGTGGAATTCGGACACGACAACA ACC-3'), F-IVSARE
(GAGACAGCTGAACCACGTGGTCAAGTGG-3'), F-MYC (5'CAGTACCA
GCTGGTCAAGTGGGCCAAGGCCTTG-3'), F-MC350 (5'-ACGTGGTCACAGC
TGCCAAGGCCTTG-3'), and F-MYCFOOT
(5'-CAGTACGAATTCCGTGGTCAAGTGGGCCAAGGCC-3') to produce the
3'-fragments of each mutant. The resulting 5'- and 3'-fragments overlap
approximately 1015 bp surrounding the mutant sequences where they
serve as template in the second round of PCR. The second round of PCR
connects the 5'- and 3'-fragments using the F-H3CAU and R-XbaCUA
primers. The resulting 350-bp fragment was cloned into the pAMP vector
using uracil DNA glycosylase. The 350-bp mutant constructs were
liberated by digestion with HindIII and XbaI and
subcloned into a HindIII/XbaI-digested pBLCAT2
vector for reporter gene assays or used for footprinting studies. The
resulting reporter gene constructs are called mARE1A-CAT, mIVSARE-CAT,
and mMyc-CAT. Each inserted 350-bp fragment was sequenced to confirm
that each construct was in the 5'-3' orientation and upstream of the tk
promoter of pBLCAT2.
Expression and Purification of AR Proteins
GST-hAR (472917) plasmid encodes a fusion protein containing
GST linked to the DNA- and hormone-binding domains of human AR protein
(GST-AR) and was provided by Dr. M. J. McPhaul (60). The
expression and purification of GST-AR were done as previously described
(4). The amino terminal-deleted AR lacking the GST moiety was obtained
essentially as described by Roehrborn et al. (60). Briefly,
purified GST-AR fusion protein was bound to glutathione Sepharose 4B
(Pharmacia Biotech, Piscataway, NJ). Column-bound
GST-AR was digested with thrombin (final concentration of 2 U/ml) in
100 mM Tris (pH 8), containing 150 mM NaCl and
2.5 mM CaCl2. Digestion proceeded for 8 h
at 20 C, followed by an additional 2 h at room temperature. The
reaction was stopped by adding EDTA to a final concentration of 5
mM; AR protein was eluted in PBS flowthrough. The purity of
the AR protein was checked by SDS-PAGE (Coomassie stain), and the
amount of AR protein was quantitated by the Bradford method
(Bio-Rad Laboratories, Inc., Richmond, CA).
Isolation of Nuclear Proteins
PC3 cells were grown to 8590% confluency, washed with
ice-cold 1xPBS, and scraped into 10 ml of PBS. Cells were centrifuged
and pellets were resuspended in 1xPBS, rapidly frozen in an ETOH/dry
ice bath, and then thawed at 37 C for 5 min. Cell extracts were
incubated on ice in Hypotonic Solution A [10 mM HEPES, pH
7.9; 10 mM KCl; 1.5 mM MgCl2; 1
mM dithiothreitol (DTT)] for 10 min. Lysis was monitored
by trypan blue exclusion. The extracts were centrifuged and the
supernatant discarded. The resulting pellet was resuspended in
Hypertonic Solution C [10 mM HEPES, pH 7.9; 0.4
M KCl; 1.5 mM MgCl2; 25% Glycerol;
0.2 mM EDTA; 1 mM DTT; 0.5 mM
phenylmethyl sulfonylfluoride (PMSF)] and shaken on ice for 30 min.
After centrifugation, the supernatant was transferred to a new tube and
diluted with Buffer D (20 mM HEPES, pH 7.9; 50
mM KCl; 20% Glycerol; 0.2 mM EDTA; 1
mM DTT; 0.5 mM PMSF). Protein concentrations
were determined by the Bradford method (Bio-Rad Laboratories, Inc., Hercules, CA) and aliquots stored at -80.
DNAse I Footprinting
The AR cDNA fragments (350WT, IVS, mARE-1, or mARE-2) were
labeled at either the 5'- or 3'-end by end-filling of
HindIII- or XbaI-linearized plasmid using Klenow
fragment and [
-32P]dCTP. The labeled fragments were
subsequently released by digestion with either XbaI or
HindIII and isolated after electrophoresis. BSA or amino
terminal-deleted AR (20 µg), GST or GST-AR fusion proteins (20 µg),
or PC3 nuclear extract or buffer control (10 or 20 µg) were incubated
with 20,000 cpm of labeled DNA fragments (
5 fmol) in 40 µl DNA
binding buffer containing 2.5 µg polyd(I-C) and 5 µg BSA. After 30
min incubation at room temperature, MgCl2 and
CaCl2 were adjusted to 4 mM and 2.5
mM, respectively. DNAse I (0.2 ng; Boehringer Mannheim) was
immediately added to the reaction mixture and the mixture was incubated
for 2 min at room temperature. The digestion reactions were stopped by
adding 40 µl inactivation buffer [20 mM Tris-HCl (pH
7.8), 20 mM EDTA (pH 8.0), 300 mM NaCl, 1%
SDS, 100 µg/ml tRNA, and 0.5 mg/ml proteinase K] and incubated at 37
C for 1 h. The sample was phenol extracted and the DNA was
precipitated with ethanol. DNA pellets were resuspended in loading
buffer [95% formamide, 20 mM EDTA (pH 8.0), 0.05%
bromophenol blue, 0.05% xylene cyanol FF] and resolved on an 8%
sequencing gel. DNA size ladder or 350-bp sequencing product was run in
parallel. The gel was then dried and subjected to autoradiography.
DNAse I footprint data were quantified by phosphorimage analysis of the
dried gels. The amount of radioactivity in each protected region
(standardized for each gel) was determined relative to a region outside
the footprint.
Gel Mobility Shift Assays
Wild-type and mutant 350-bp fragments were obtained by digestion
of their respective CAT vectors (350WT-CAT, mARE1A-CAT, mIVSARE-CAT,
mMyc-CAT) with HindIII and XbaI, followed by
purification from agarose gels. GST, affinity-purified GST-AR fusion
proteins (500 ng), or PC3 nuclear extract proteins (5 µg) were mixed
with 5 µg polyd(I-C) (from Roche Molecular Biochemicals), 1 µg BSA, and 20,000 cpm
32P-labeled 350-bp fragment (
2.5 fmol) in DNA binding
buffer [10 mM Tris-HCl (pH 7.9), 50 mM KCl,
10% glycerol, 0.2 mM EDTA (pH 8.0), 1 mM DTT,
and 50 nM R1881] to a final volume of 20 µl, and
incubated at room temperature for 20 min. For the competition studies,
the proteins were preincubated for 20 min at room temperature with the
indicated unlabeled 350-bp DNA fragment competitor (50-, 100-, 250-fold
molar excess) or Myc oligonucleotide competitor (25-, 50-fold molar
excess), followed by addition of the labeled 350WT probe. The sequences
for the Myc competitor oligonucleotides were
5'-GGAAGCAGACCAC-GTGGTCTGCT-TCC-3' (consensus site
is boldface) and 5'-GGAAGCAGACCAGCTGGTCTGCTTCC-3'
(mutated consensus site is boldface). The samples were
loaded directly onto a prerun, nondenaturing 5% polyacrylamide gel
(acrylamide-bis, 29:1) in 45 mM Tris-borate (pH 8.0), 1
mM EDTA (pH 8.0), run at 200 V for 1 h and 250 V for
23 h at room temperature, and then dried onto a filter paper and
visualized by autoradiography. The DNA-protein complexes were
quantitated by PhosphorImager Analysis using ImageQuant software
(Molecular Dynamics, Inc., Sunnyvale, CA).
For the gel shift-based Western blot, a preparative gel shift using 100
µg of PC3 nuclear extracts was incubated under the above conditions
and separated on a 5% nondenaturing gel. The gel was run longer (1.5 h
at 250 V and 4 h at 200 V) to further separate and distinguish the
nuclear protein-DNA complexes. The gel was UV cross-linked (1200
joules) and exposed to film to visualize the complexes. The indicated
bands and regions were cut out and incubated at room temperature in 5x
Laemmli loading buffer for 20 min. The gel slices and buffer were
loaded into wells of an SDS-PAGE gel (13% resolving/4% stacking) and
separated. Complexes were transferred to nitrocellulose membrane and
processed for immunoblotting using standard procedures. Briefly,
filters were incubated in blocking solution (5% dry milk, 1% Tween)
for 1 h followed by incubation with primary antibody (anti-Max
sc-765, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or
anti-Myc panMyc (a generous gift from Dr. T. Littlewood) for 1 h.
After washing, the blot was incubated with horseradish
peroxidase-conjugated secondary antibody (antirabbit, Santa Cruz Biotechnology, Inc.), and proteins were visualized using the ECL
system (Amersham Pharmacia Biotech, Buckinghamshire, UK)
following the suppliers instructions.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Marco Marcelli for providing AR cDNA clones, Dr.
Michael McPhaul for the GST-AR (472917) construct, Dr. Bruno Amati
for advice and providing the pBJ3, Myc, Max, MycRx, and MaxRx vectors,
and Dr. Trevor Littlewood for the pan-Myc antibody. We are grateful to
Dr. Bonnie Blomberg and Ms. Carol Maiorino for helpful comments on the
manuscript, Dr. Olga Hernandez for suggestions on gel
shift/immunoblots, and to Dr. Melanie Palmer for advice on cloning
strategies.
 |
FOOTNOTES
|
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Address requests for reprints to: Kerry L. Burnstein, Ph.D., Department of Molecular and Cellular Pharmacology (R-189), P.O. Box 016189, Miami, Florida 33101. e-mail: kburnste@chroma.med.miami.edu.
This work was supported by NIH Grant DK-45478 to K.L.B. J.M.G.
received support from the American Heart Association-Florida Affiliate
(Fellowship 9604012) and NIH Grant T32 HL-07188.
1 Current Address: Department of Pathology, Johns Hopkins University,
Baltimore, Maryland 21205. 
2 Current Address: Department of Pediatrics, University of Miami
School of Medicine, Miami, Florida 33101. 
Received for publication October 28, 1998.
Revision received July 20, 1999.
Accepted for publication July 23, 1999.
 |
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