New Androgen Response Elements in the Murine Pem Promoter Mediate Selective Transactivation
Karina Barbulescu,
Christoph Geserick,
Iris Schüttke,
Wolf-Dieter Schleuning and
Bernard Haendler
Research Laboratories of Schering AG,
D-13342 Berlin,
Germany
Address all correspondence and requests for reprints to: Dr. Bernard Haendler, Experimental Oncology, Schering AG, D-13342 Berlin, Germany. E-mail: bernard.haendler{at}schering.de
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ABSTRACT
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The Pem homeobox transcription factor is expressed under androgen
control in the testis and epididymis. It is also transcribed in the
ovary, muscle, and placenta. The mouse Pem gene promoter was cloned and
sequenced. It was analyzed in transactivation tests using CV-1 and PC-3
cells expressing the AR and found to be strongly stimulated by
androgens. EMSAs and mutational analysis of the Pem promoter allowed
the identification of two functional androgen response elements named
ARE-1 and ARE-2. They both differed from the consensus semipalindromic
steroid response element and exhibited characteristics of direct
repeats of the TGTTCT half-site. Unlike the steroid response element,
both Pem androgen response elements were selectively responsive to
androgen stimulation. Specific mutations in the left half-site of Pem
ARE-1 and ARE-2, but not of the steroid response element, were still
compatible with AR binding in the EMSA. In addition, Pem ARE-1, but not
ARE-2 or the steroid response element, showed some flexibility with
regard to spacing between half-sites. These results strongly suggest
that the AR interacts differently with direct repeats than with
inverted repeats, potentially leading to cis
element-driven selective properties. Thus, the existence of several
classes of DNA response elements might be an essential feature of
differential androgen regulation.
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INTRODUCTION
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THE SEMIPALINDROMIC STEROID response
element (SRE) mediates AR function in several androgen-dependent
target genes (1, 2, 3). This cis-acting
element is also recognized by the GR, the PR, and the MR, all of which
closely resemble the AR in their DNA-binding regions, especially in the
P- and D-boxes (2, 4). Mutational analysis of steroid
receptors identified amino acids of the first zinc finger
-helix
that establish contact with specific nucleotides of the DNA element and
other residues that prevent the receptor from binding to improper sites
(5, 6).
Comprehensive in vitro selection procedures have shown the
optimal androgen response element (ARE) to be virtually identical to
the SRE (7, 8). One study has reported the identification
of a novel selective element that, as yet, has not been found in
naturally occurring promoters (9). All of these results
are based on highest DNA-binding affinity as the selection step, which
is not necessarily the primary property by which gene control is
achieved in vivo. Indeed, only the suboptimal binding sites
may allow specificity for a given steroid receptor by preventing
recognition by other receptors (10). A survey of natural
functional AREs shows that they generally vary from the consensus SRE
and display less binding affinity to the AR (3, 11).
Consequently, several such elements are frequently present in
androgen-regulated genes and may be necessary for full-scale
stimulation (12, 13, 14, 15, 16).
Specific steroid hormone action can be mediated through nonselective
DNA elements in a variety of ways. The levels of receptor and hormone
available in a given cell or tissue may play important roles (17, 18). Another mechanism is provided by differential chromatin
remodeling, as documented for the AR and GR, using the mouse mammary
tumor virus promoter (19). Several examples in which the
interplay of a steroid receptor with other transcription factors
accounts for discriminating effects have also been reported (13, 20, 21). The preferential interaction with cofactors represents
a further possibility, but only a few specific cofactors have been
identified (22, 23). Indeed, most of them enhance the
activity of several steroid receptors and have a broad tissue
distribution (24, 25). Finally, cooperation among weak
SREs and with auxiliary elements might lead to selective stimulation,
as shown for the sex-limited protein (Slp), the probasin (PB), the
prostate-specific antigen, and the 20-kDa protein genes (12, 13, 14, 15, 26, 27, 28). Studies of the Slp promoter demonstrate that
interactions between specific AR regions are essential to enhance
cooperativity at suboptimal DNA-binding elements (26).
Very recently, it has become apparent that unique variations within the
DNA-binding sequence may have a dramatic impact on the recognition by a
given nuclear receptor. Thus, response elements displaying androgen
vs. glucocorticoid selectivity have been identified in the
promoter of the PB, the secretory component, and the Slp genes
(10, 29, 30). They display variations of the TGTTCT
half-site with direct repeats seeming to favor preferential AR binding
(10, 29). A detailed mutational analysis of the AR has
shown the second zinc finger and a C-terminal extension to be
implicated in the differential recognition of such direct repeats
(31, 32). On the other hand, a perfect repeat of the
TGTTCT motif spaced by nine nucleotides is recognized by the GR
(33).
The Pem homeobox gene is mainly expressed in reproductive organs, in
the muscle, and in the placenta (34). A detailed analysis
of the rat gene has shown that two different promoters are used. The
proximal promoter is androgen dependent and controls expression in the
epididymis and testis, and the distal promoter is androgen independent
and responsible for expression in the testis, ovary, muscle, and
placenta (34). In the testis, Pem is expressed in Sertoli
cell nuclei, suggesting an important role in spermatogenesis
(35, 36, 37).
Here we describe the molecular mechanisms underlying the regulation of
the murine Pem gene. We demonstrate that the Pem promoter is
selectively stimulated by androgens. It contains two functional AREs,
ARE-1 and ARE-2, which differ in sequence from the classic SRE. Both
exhibit characteristics of direct repeats and are preferentially
stimulated by androgens. Mutational analysis strongly suggests a novel
mode of interaction between the direct repeat Pem AREs and the AR.
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RESULTS
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The Pem Upstream Region Is Conserved between Mouse and Rat
The upstream region of the mouse Pem gene was amplified by nested
PCR using antisense primers specific to the 5'-end of the transcribed
part and sense primers corresponding to the adaptors ligated to the
genomic DNA fragments used as template. After subcloning, the DNA
sequence was determined on fragments originating from three separate
amplifications. Two clones were identical over 1,100 bp of upstream
region, whereas the third clone showed a single nucleotide exchange (a
G instead of an A at position -93). This position is also occupied by
an A in a sequence found later in a public database (accession number
U50747).
A comparison between the mouse and rat Pem upstream regions showed a
high sequence conservation, especially in the part previously defined
as the proximal promoter (34) between positions -444 and
-1, where an identity of 88% was calculated. Both sequences were
largely colinear except for a 3-nucleotide deletion upstream of
position -388 and an 11-nucleotide insertion upstream of position
-275 in the mouse sequence. The region corresponding to the
muscle-specific exon in the rat immediately upstream of the proximal
promoter displayed only 69% sequence conservation compared with the
mouse, whereas the second exon between positions -681 and -626 was
82% identical. A conservation of 70% was found for the intron lying
between these two exons. The sequence upstream of exon 2 is only
partially characterized in the rat, so a meaningful comparison with the
mouse counterpart was not possible.
Putative cis-Acting Elements of the Mouse Pem
Promoter
A search for putative regulatory elements was carried out in the
murine Pem upstream region (Fig. 1
). No
obvious SRE could be found even though two TGTTCT half-sites were
located starting at positions -739 and -725. However, they are
upstream of the proximal promoter shown to be responsible for androgen
control in the rat, and no sequence resembling a second half-site was
found in the vicinity. When allowing for variations in the consensus
SRE, three candidates could be found in the proximal promoter:
GGCACCctaAGTTCT, AGCACAtcgTGCTCA, and AGATCTcattcTGTTCC, starting at
positions -299, -247, and -85, respectively. All contained two
variant copies of the canonical SRE half-site, including the G and C
contact nucleotides, and were spaced by three or five nucleotides.
These elements were further analyzed (see below).

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Figure 1. Nucleotide Sequence of the Mouse Pem Promoter
The putative upstream exon regions are indicated in capital
letters, as is the translated region of the first epididymal
exon. Numbering starts at the translation initiation
codon. ARE-1 and ARE-2 are boxed, and the nonbinding
ARE-like motif is indicated with a broken line. Two
TGTTCT havf-sites are also shown with a broken line. The
potential initiator regions and the motifs matching transcription
factor binding sites are underlined.
Arrowheads indicate the beginning of the two promoter
constructs analyzed in transactivation assays. GenBank accession
number: AF410462.
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As previously noted in the rat, the mouse Pem gene has no TATA
box. Several stretches resembling the initiator often used in TATA-less
genes (38) were found in the upstream region (Fig. 1
). In
addition, motifs with a perfect match to several other reported
DNA regulatory elements were identified in one or the other
orientation. Using the PatSearch tool (39), consensus
binding sites for Yi, CP2, LIM-only protein-2 (Lmo2), activator
protein-2 (AP-2), hepatocyte nuclear factor-4 (HNF4), TCF11,
Ets/polyoma virus enhancer A binding protein-3 (PEA3), ying-yang-1
(YY1), octamer-binding protein-1 (Oct-1), and the GATA factor
were found (Fig. 1
).
Selective Stimulation of the Pem Promoter by Androgens
Two Pem reporter plasmids were devised. For the longer wild-type
construct, the upstream region from position -1,139 (Fig. 1
), just
downstream of a TG dinucleotide repeat and covering exon 2 and the
muscle-specific exon, to position -36 was placed upstream of the
luciferase reporter gene. For the shorter, proximal promoter construct,
a fragment starting at position -444, downstream of the
muscle-specific exon, and ending at position -36 was similarly
introduced into the reporter vector.
CV-1 cells were used for the transfections because they do not express
endogenous steroid receptors (40, 41). The Pem promoter
constructs were cotransfected with expression vectors for the AR, GR,
or PR and treated with the appropriate steroid (Fig. 2
). For both promoter constructs, the
strongest effects were noted after adding the androgen R1881. Less
stimulation was seen after progestin treatment, and only a small effect
was observed after glucocorticoid treatment. The inductions were
altogether somewhat higher when using the shorter construct.

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Figure 2. Selective Androgen Stimulation of the Pem Promoter
Pem gene upstream regions starting at position -1139 or -444 were
introduced into the pGL3-Basic plasmid in front of the luciferase
reporter gene. These constructs were transfected into CV-1 cells
transiently expressing the AR, GR, or PR, as indicated. Hormonal
treatment was with 10-9 M R1881, dexamethasone
(Dex), or R5020. The luciferase activity was measured after 23 h.
The results are representative of three separate experiments, and the
bars indicate means ± SEM of
sextuplicate values. The fold stimulation in the presence of the added
steroid is given.
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Specific Binding of the -247 and -85 Motifs to the AR
The EMSA and in vitro translated full-length human AR
were used to analyze putative response elements in the Pem promoter. We
found that the -85 and -247 elements, but not the -299 motif,
displayed binding to the AR. They were named ARE-1 and ARE-2,
respectively. The complexes formed were less abundant than the complex
between the SRE and the AR, and they migrated at about the same level.
In the case of ARE-1, binding to the AR was competed by both the
cognate sequence and the SRE, demonstrating AR specificity (Fig. 3A
, lanes 9 and 10). A mutated SRE, in
which the G and C contact nucleotides had been mutated, making it
unable to bind to the AR, did not compete for complex formation (lane
11). A supershift was observed using an AR-specific antibody, further
demonstrating specificity (lane 12). When analyzing the control SRE, we
found strong autocompetition by the cognate element but only weak
competition by ARE-1 (lanes 3 and 4). Again, the mutated SRE did not
compete (lane 5). A supershift was observed in the presence of an
anti-AR antibody (lane 6). Similar results were obtained with ARE-2,
i.e. autocompetition by the same sequence and
cross-competition by the SRE (Fig. 3B
, lanes 7 and 8). In addition,
ARE-2 was able to efficiently compete for the formation of SRE/AR
complexes (lane 4). Specificity was shown using an anti-AR antibody
to supershift the complex (lanes 5 and 9). No specific complex was
formed when ARE-2 was incubated with an unprogrammed extract (see Fig. 8
).

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Figure 3. Binding of Pem AREs to the AR
Double-stranded SRE, ARE-1, or ARE-2 oligonucleotides were labeled with
DIG and incubated with in vitro translated (i.v.tr.)
human AR. The DNA/AR complexes were separated on a 5% polyacrylamide
gel, blotted onto a nylon membrane, and detected by a specific anti-DIG
alkaline phosphatase antibody. The chemiluminescence CSPD-Star
substrate was used for detection. Unprogrammed reticulocyte lysate was
used in the control (Co). Molar excesses of cold SRE, non-AR binding
mutated SRE (SRE-mut), ARE-1, or ARE-2 oligonucleotides were added as
indicated. The supershift obtained in the presence of a specific
anti-AR antibody is indicated by asterisks. A,
Comparison between ARE-1 and the SRE. B, Comparison between ARE-2 and
the SRE.
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Figure 8. Mutational Analysis of ARE-2
A, DNA sequences of the ARE-2 mutants analyzed. The changes from the
original sequence are underlined. nt, Nucleotide. B,
EMSA of ARE-2 mutants. Double-stranded oligonucleotides were labeled
with DIG and incubated with in vitro translated human
AR. The DNA/AR complexes were separated on a 5% polyacrylamide gel,
blotted onto a nylon membrane, and detected with a specific anti-DIG
alkaline phosphatase antibody. The chemiluminescence CSPD-Star
substrate was used for detection. A representative of three separate
experiments is shown. C, The amount of DNA/AR complex was quantified
using the analysis software of the ChemiImager.
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ARE-1 and ARE-2 Mediate the Androgen Response of the Pem
Promoter
ARE-1 and ARE-2 were individually changed to irrelevant sequences
in the proximal Pem promoter construct by site-directed mutagenesis.
The reporter plasmids were first analyzed in CV-1 cells transiently
expressing steroid receptors (Fig. 4A
).
In the presence of AR, a strong up-regulation by the cognate ligand was
seen using the wild-type Pem promoter. This effect was markedly reduced
for both mutated forms. The ARE-2 mutant was down to 40% and the ARE-1
mutant was down to 65% of wild-type-induced levels. In a parallel
experiment using CV-1 cells transiently expressing the GR, the
wild-type Pem promoter was only moderately stimulated by glucocorticoid
treatment. Mutation of ARE-1 and even more so of ARE-2 severely
decreased the response.

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Figure 4. Mutational Analysis of the Pem Promoter
Reporter constructs containing the Pem proximal promoter as wild-type
form (WT) or with mutated ARE-1 (Mut ARE-1) or mutated ARE-2 (Mut
ARE-2) were used for transfection. Luciferase activity was measured as
sextuplicate values after 23 h. The results show the fold
induction determined in the presence of ligand and are representative
of three separate experiments. A, CV-1 cells were cotransfected with
the reporter vector and with an expression vector for the AR or GR, as
indicated. Stimulation was with 10-9 M R1881
or dexamethasone (Dex). B, PC-3/AR cells were transfected with the
reporter vector. Stimulation was with 10-9 M
R1881.
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To determine whether these results could be extended to other
cell lines and to minimize variations linked to transient expression
systems, we also transfected the reporter constructs into a PC-3 cell
line stably expressing the human AR (PC-3/AR). A robust androgen
stimulation was noted for the wild-type construct, but not as
pronounced as when using CV-1 cells (Fig. 4B
). Again, mutation of ARE-1
and especially of ARE-2 diminished the effects of androgens.
ARE-1 and ARE-2 Display Cooperativity in AR Binding
Because the mutations of ARE-1 and ARE-2 had different effects on
stimulated Pem promoter activity, we looked at possible cooperative
interactions between these elements. We devised probes containing two
response elements: ARE-1/ARE-1, ARE-2/ARE-2, or ARE-2/ARE-1. The
interaction with the AR was analyzed in the EMSA using nuclear extracts
prepared from CV-1 cells transfected with an AR-encoding plasmid and
treated with R1881. The expression of AR in nuclear extracts, but not
in cytoplasmic extracts or in fractions from untransfected cells, was
shown by Western blot analysis using a specific antibody (not shown).
In the EMSA, more DNA/AR complex was formed with the ARE-2/ARE-1 probe
than with the ARE-1/ARE-1 probe or the ARE-2/ARE-2 probe (Fig. 5
, lanes 57). No specific complex was
formed when using protein extracts from mock-transfected CV-1 cells
(lanes 24).

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Figure 5. Cooperativity of Pem AREs
Double-stranded ARE-1/ARE-1, ARE-2/ARE-2, or ARE-2/ARE-1
oligonucleotides were labeled with DIG and incubated with 6 µg of
nuclear extracts prepared from CV-1 cells transfected with an AR
expression vector (+ pSG5/AR) or with an empty vector (+ pSG5). The
DNA/AR complexes were separated on a 5% polyacrylamide gel, blotted
onto a nylon membrane, and detected by a specific anti-DIG alkaline
phosphatase antibody. The chemiluminescence CSPD-Star substrate was
used for detection.
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ARE-1 and ARE-2 Are Differentially Stimulated by Androgens,
Glucocorticoids, and Progestins
Next, we examined the properties of Pem ARE-1 outside of its
promoter context, placed as one to four copies in the thymidine kinase
(TK)-luciferase reporter plasmid. Upon cotransfection of CV-1
cells with an expression vector for the AR, strong induction was
observed after R1881 treatment when two or four elements were present
(Fig. 6A
). In comparison, cotransfection
with a GR expression vector and treatment by the cognate ligand gave a
stimulation less than half as pronounced. The response to the progestin
R5020 was intermediate. Conversely, the SRE responded highly to
glucocorticoids and to progestins and far less to androgens (Fig.
6A). Altogether, the results demonstrate that ARE-1 was far more
responsive than the SRE to androgen stimulation and less responsive to
progestin and especially to glucocorticoid treatment.

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Figure 6. Selectivity of Pem AREs
A, One to four copies of ARE-1 or the SRE were introduced into the
pTATA plasmid upstream of the TK minimal promoter and of the luciferase
reporter gene. The reporter constructs were cotransfected into CV-1
cells together with an expression plasmid for the AR, GR, or PR.
Hormonal treatment was with 10-9 M of the
cognate ligand, as indicated. Luciferase activity was measured after
23 h. The results are representative of at least three separate
experiments, and the bars indicate means ±
SEM of sextuplicate values. The fold stimulation in the
presence of the ligand is given. B, One to four copies of ARE-2 or the
SRE were introduced into the pTATA plasmid upstream of the TK minimal
promoter and of the luciferase reporter gene. The reporter constructs
were cotransfected into CV-1 cells together with an expression plasmid
for the AR, GR, or PR. Hormonal treatment was with 10-9
M of the cognate ligand, as indicated. Luciferase activity
was measured after 23 h. The results are a representative of at
least three separate experiments, and the bars indicate
means ± SEM of sextuplicate values. The fold
stimulation in the presence of the ligand is given.
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The steroid selectivity of Pem ARE-2 was investigated by performing
similar transactivation experiments in CV-1 cells (Fig. 6B
). One to
four copies of ARE-2 were placed in the TK-luciferase reporter plasmid
as described above. Upon cotransfection with the appropriate steroid
receptor expression plasmid, ARE-2 present as one, two, or three copies
yielded a stronger response to R1881 than to dexamethasone or R5020
(Fig. 6B
). No significant difference was seen with four ARE-2 copies.
Compared with the SRE, ARE-2 was more responsive to androgen but less
responsive to glucocorticoid. Concerning progesterone vs.
androgen effects, less induction was noted for ARE-2, except when four
copies were tested (Fig. 6B
).
Specific Mutations in ARE-1 and ARE-2 Are Still Compatible with AR
Binding
Because the Pem AREs deviate from classic SREs with regard to the
configuration of their half-sites (Table 1
), we carried out a comparative
mutational analysis to determine which nucleotides were important for
recognition by the AR. We first analyzed ARE-1. The mutants were
analyzed by the EMSA using the full-length AR (Fig. 7
, A and B), and the amount of complex
formed was quantified (Fig. 7C
). All of the complexes formed migrated
at the same level, showing that only receptor homodimers bound to the
DNA elements. Mutations of the G and C contact nucleotides in both
half-sites (M1) or in the right half-site only (M3) were not compatible
with complex formation. Conversely, the same mutations in the left
half-site only (M2) increased AR binding to the element. The strongest
complexes were observed when introducing a G nucleotide in the left
half-site to nearly reconstitute a consensus SRE with inverted repeat
features (M6 and M7). Reduction of the spacer from five to three
nucleotides (M4 and M5) resulted in increased complex formation,
whereas a change to one, two, or six nucleotides (M9, M8, and M10) was
incompatible with AR binding.

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Figure 7. Mutational Analysis of ARE-1
A, DNA sequences of the ARE-1 mutants analyzed. The changes from the
original sequence are underlined. nt, Nucleotide. B,
EMSA of ARE-1 mutants. Double-stranded oligonucleotides were labeled
with DIG and incubated with in vitro translated human
AR. The DNA/AR complexes were separated on a 5% polyacrylamide gel,
blotted onto a nylon membrane, and detected with a specific anti-DIG
alkaline phosphatase antibody. The chemiluminescence CSPD-Star
substrate was used for detection. A representative of three separate
experiments is shown. C, The amount of DNA/AR complex was quantified
using the analysis software of the ChemiImager.
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Next, we examined ARE-2. Similar changes were introduced into this
element (Fig. 8A
), and binding by the AR
was analyzed as described above with the EMSA (Fig. 8B
) and quantified
(Fig. 8C
). Mutations of the G and C contact nucleotides in both
half-sites (M1) or just in the right half-site (M3) almost entirely
prevented the formation of a complex with the AR. Conversely, the
equivalent mutations in the left half-site (M2) did not affect AR
binding. Changes in the size of the spacer to one, two, four, or five
nucleotides were followed by a loss of AR binding (M4M7).
Finally, the effect of comparable changes in the SRE was assessed (Fig. 9
). Here, mutations of the G and C
contact nucleotides in one or the other half-site were both detrimental
to complex formation with the AR (M1M3). Any change in the spacing
brought about a complete loss of AR binding (M4M9).

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Figure 9. Mutational Analysis of the SRE
A, DNA sequence of the SRE mutants analyzed. The changes from the
original sequence are underlined. nt, Nucleotide. B,
EMSA of SRE mutants. Double-stranded oligonucleotides were labeled with
DIG and incubated with in vitro translated human AR. The
DNA/AR complexes were separated on a 5% polyacrylamide gel, blotted
onto a nylon membrane, and detected with a specific anti-DIG alkaline
phosphatase antibody. The chemiluminescence CSPD-Star substrate was
used for detection. A representative of three separate experiments is
shown. C, The amount of DNA/AR complex was quantified using the
analysis software of the ChemiImager.
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DISCUSSION
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Pem gene expression is regulated by androgens in the epididymis
and testis. We cloned the murine Pem promoter and analyzed it in two
different cell lines by transactivation tests. A strong androgen
response was observed in CV-1 cells and in PC-3 cells transfected
transiently or constitutively with the AR. These effects were highly
selective, as progestins and especially glucocorticoids elicited far
less response in CV-1 cells cotransfected with the corresponding
receptor. This is in line with the in vivo situation, in
which the Pem gene is mainly transcribed in the testis and epididymis
under androgen control, and in the ovary, for which no data about
progesterone regulation are available. The preferential stimulation by
R1881 suggested the presence of androgen-selective DNA response
elements in the mouse Pem promoter. Sequence analysis revealed the
presence of motifs related to AREs, two of which were found to form
specific complexes with the AR in the EMSA. They were entirely
conserved in the rat Pem promoter, suggesting an important regulatory
role. The functionality of these elements was confirmed in
transactivation studies in CV-1 and PC-3/AR cells. Alteration of ARE-1
or ARE-2 led to a marked loss of the androgen response of the Pem
promoter. Because the ARE-2 mutation had more impact, it is probably
the main player in the androgen control of the Pem promoter, in line
with its stronger AR-binding properties in the EMSA as a single
element. The additional presence of ARE-1, however, appeared necessary
to yield the full response, suggesting cooperative interactions between
both elements. The effect is most apparent in PC-3/AR cells, in which
ARE-1 conveys poor androgen response in the context of the Pem promoter
but enhances 2-fold that of ARE-2. Cooperativity is further
corroborated by the stronger signal observed in the EMSA for the AR
complexed to the ARE-2/ARE-1 probe compared with the ARE-1/ARE-1 and
ARE-2/ARE-2 probes. Interestingly, the ARE-2 tandem element exhibited
weaker complex formation than the ARE-1 tandem element, whereas the
opposite effect was observed with single elements. These data support
the notion that synergy between weak AREs is the basis for a strong,
selective androgen response (26). It is also notable that
ARE-1 overlaps a potential initiator element, which might have
implications for how Pem expression is controlled in vivo.
In fact, the Pem gene promoter is one of the very few that is both
androgen regulated and TATA-less. Another example is the AR gene
promoter itself, which is transcriptionally up- or down-regulated by
androgens, depending on the cell context (42, 43, 44).
Transactivation assays showed the Pem AREs to exhibit different
response profiles to steroids compared with previously described AREs.
None showed the high glucocorticoid selectivity of the consensus SRE
attributed to a stronger affinity of the GR DNA-binding domain and to a
higher dissociation rate of the AR for this element (18).
Because many of the response elements described so far in
androgen-regulated genes correspond to this consensus, it is
no surprise that they are not selective for androgens (1, 6, 45).
An alignment of the Pem elements with the consensus SRE shows
that they do not fit the classic inverted repeat with a
three-nucleotide spacing model (Table 1
). Pem ARE-1 is composed of two
half-sites, including the G and C contact bases, but separated by five
nucleotides. This arrangement may be seen as either a direct or an
inverted repeat. However, because only inverted repeats with a
three-nucleotide spacer are bound by the AR (Fig. 9
), it is likely that
ARE-1, with its five-nucleotide spacer, is recognized as a direct
repeat. This might explain the preferential response of ARE-1 to
androgens. A feature probably essential for androgen selectivity is the
T nucleotide present at position -4, because the complementary A
nucleotide on the other strand has recently been shown to exclude GR
interaction (46). The molecular basis for androgen
vs. progesterone selectivity is less clear, but the
stimulation of ARE-1 by R5020 is in line with the expression of Pem in
the ovary (34) and the response of its promoter to
progestins (Fig. 2
).
ARE-2 is composed of two half-sites spaced by three nucleotides
in which four of six possible positions are maintained in the direct
repeat mode (Table 1
). It displayed androgen selectivity when using one
to three copies. When testing four copies, similar induction values
were measured for androgen, glucocorticoid, and progesterone treatment,
probably as a result of a limiting factor in the cells. This was also
apparent from the response saturation observed when comparing three and
four ARE-2 copies in the androgen-treated cells or two and four SRE
copies in the dexamethasone-treated cells. Altogether, these results
indicate that both ARE-1 and ARE-2 are implicated in the selective
androgen response of the Pem promoter.
Our mutational analysis showed only the right half-site of Pem ARE-1
and ARE-2 to be essential for AR binding, whereas both half-sites of
the SRE were important. This result strongly suggests a two-step
recognition mechanism by the AR in which prior binding of the right
half-site of the DNA element is a prerequisite for the recognition of
the left half-site. This implies that the right half-site is stronger
with regard to AR binding, which has also been found for the PB direct
repeat (32). Another implication is that the orientation
of a direct repeat in its promoter context may be of much more
significance than that of the more symmetrical inverted repeat with
regard to interaction with other transcription factors and cofactors.
The model is also compatible with less symmetry in the AR homodimer,
which might not necessarily bind to the Pem AREs in the classic
head-to-head configuration (5). Indeed, the fact that for
ARE-1, spacings of three and five nucleotides both allow binding by the
AR, whereas no flexibility is possible for the SRE, suggests a novel
arrangement of the AR homodimer bound to this sequence. This might be
in the head-to-tail configuration, as suggested in the case of the PB
direct repeat element (32). Mutational analysis found
three amino acids of the second zinc finger and the hinge region that
were implicated in selective binding. For Pem ARE-1, additional domains
located in the N or C terminus of the AR may also be involved, because
the longer spacing between both half-sites might prohibit close
contacts of the zinc finger and hinge regions. Crystallographic studies
of the GR using an inverted repeat element with three- or
four-nucleotide spacers clearly demonstrate the spatial constraints
prohibiting contacts between the dimerization interface of the
head-to-head GR central region (5). The essential role of
a dimerization domain located in the ligand-binding domain of the GR
for binding to a direct repeat with a nine-nucleotide spacer has been
reported (33). Finally, the fact that direct repeats are
functional AREs opens up the possibility that heterodimers between the
AR and other nuclear receptors may recognize such elements, in analogy
to heterodimers formed by the RXR on other direct repeat elements
(47).
In conclusion, we have analyzed the molecular mechanisms responsible
for the androgen control of the Pem gene. Two new AREs with direct
repeat features and novel selectivity profiles have been identified.
Because direct repeat elements have altogether a weaker affinity for
the AR than inverted repeats, the mere local hormone concentration in
tissues may represent a first selectivity step for androgen
vs. glucocorticoid stimulation in vivo. In
addition, direct repeat elements may be recognized by the AR dimer in a
head-to-tail rather than a head-to-head configuration, as shown for
palindromic elements. This has important implications for the
intermolecular and intramolecular domain interactions taking place for
the AR and known to be essential for selective function
(26). It also implies that the set of cofactors recruited
may differ depending on the class of DNA elements recognized. Whether
ligands that specifically recognize the AR bound to various response
elements exist is currently being investigated, and this may open new
opportunities in several therapeutic areas.
 |
MATERIALS AND METHODS
|
---|
Chemicals and Cell Culture Media
The progestin R5020 was synthesized in house. R1881
(methyltrienolone) was from NEN Life Science Products
(Boston, MA), and dexamethasone was from Sigma (St. Louis,
MO). RPMI 1640, MEM, OPTI-MEM, streptomycin, penicillin, Geneticin, and
L-glutamine were obtained from Life Technologies, Inc. (Gaithersburg, MD). FCS was from PAA, Linz,
Austria). The oligonucleotides were purchased from MWG Biotech (Ebersberg, Germany) or from Carl Roth GmbH
(Karlsruhe, Germany). The digoxigenin (DIG) labeling kit, Pefabloc
SC, and FuGene 6 were from Roche Molecular Biochemicals
(Indianapolis, IN).
Cloning of Mouse Pem Upstream Region and DNA Analysis
The Mouse GenomeWalker kit (CLONTECH Laboratories, Inc., Palo Alto, CA) was used. The 5'-GTTCTTCCGAGTCTTCCTTGAC-3'
and 5'-AGGCGGAGTAGCCTGGTGAC-3' oligonucleotides were taken as reverse
primers GSP1 and GSP2, respectively. The amplification products were
separated on a 1.5% agarose gel, purified using the Silica Spin
Fragment DNA kit (Biometra, Göttingen, Germany), and cloned into
the pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA).
Sequencing was performed with Taq polymerase using the
BigDye Terminator Cycle Sequencing kit (Perkin Elmer Applied Biosystems, Foster City, CA). The amplified products were
purified from the dye terminators using Centriflex gel filtration
cartridges (MoBiTec, Göttingen, Germany) and analyzed on an ABI
PRISM 310 Genetic Analyzer (Perkin Elmer). The sequences
of both strands were determined. The GCG Software (Genetics Computer Group, Madison, WI; Ref. 48) and the
PatSearch Tool (GBF-Braunschweig; Ref. 39) were used for
DNA sequence analyses.
Plasmids
For the Pem promoter constructs, the -1,139 to -36 fragment or
the -444 to -36 fragment was PCR amplified using Taq
polymerase (Perkin Elmer) while adding the appropriate
restriction sites and introduced between the NheI and
HindIII sites of the pGL3-Basic plasmid (Promega Corp., Madison, WI). For the response element reporter
constructs, one to four copies were placed upstream of the TK minimal
promoter and of the luciferase gene by ligating the appropriate
oligonucleotides into the pTATA vector (49). They
contained the 5'-AGATCTCATTCTGTTCC-3' (Pem ARE-1),
5'-AGCACATCGTGCTCA-3' (Pem ARE-2), or 5'-GGTACATCTTGTTCA-3'
(CRISP-11253 ARE; 50) sequence flanked by the
appropriate number of bp to generate a spacing of 12 between elements.
Site-directed mutagenesis was carried out using the QuikChange kit
(Stratagene, La Jolla, CA) according to the
manufacturers instructions. DNA sequencing was performed as described
above.
In Vitro Translation
Human AR cDNA was transferred into the pCRII-TOPO plasmid
(Invitrogen). In vitro translation was carried
out with 1 µg of plasmid using the TNT T7/SP6 Coupled Reticulocyte
Lysate system and the SP6 RNA polymerase, according to the
manufacturers instructions (Promega Corp.). The level of
AR synthesized was assessed by Western blot analysis using the sc-7305
antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA)
and according to standard procedures. The reaction products were stored
at -70 C in small aliquots.
EMSA
The following oligonucleotides and their complementary strands
were used to analyze wild-type DNA elements: -85 ARE-1,
5'-CATCACAGATCTCATTCTGTTCCCGGGGAC-3'; -247 ARE-2,
5'-TCTTGCAAGCACATCGTGCTCATTACA-3'; -299 motif,
5'-TAACTGGGCACCCTAAGTTCTGCACAC-3'.
The following oligonucleotides and their complementary strands were
used to analyze cooperativity of DNA elements: ARE-1/ARE-1,
5'-CATCACAGATCTCATTCTGTTCCCGGGGACATCACAGATCTCATTCTGTTCCCGGGGA-3';
ARE-2/ARE-2,
5'-CTTGCAAGCACATCGTGCTCATTACATCTTGCAAGCACATCGTGCTCATTACA-3';
ARE-2/ARE-1,
5'-CTTGCAAGCACATCGTGCTCATTACATCATCACAGATCTCATTCTGTTCCCGGGGA-3'.
The following oligonucleotides and their complementary strands were
used to analyze mutants of ARE-1: M1,
5'-CATCACATATATCATTCTTTTACCGGGGAC-3'; M2,
5'-CATCACATATATCATTCTGTTCCCGGGGAC-3'; M3,
5'-CATCACAGATCTCATTCTTTTACCGGGGAC-3'; M4,
5'-CATCACAGATCTTTCTGTTCCCGGGGAC-3'; M5,
5'-CATCACAGATCTCATTGTTCCCGGGGAC-3'; M6,
5'-CATCACAGAGCTCATTCTGTTCCCGGGGAC-3'; M7,
5'-CATCACAGAGCACATTCTGTTCCCGGGGAC-3'; M8,
5'-CATCACAGATCTCATGTTCCCGGGGAC-3'; M9,
5'-CATCACAGATCTTTGTTCCCGGGGAC-3'; M10,
5'-CATCACAGATCTCATTCTTG-TTCCCGGGGAC-3'.
The following oligonucleotides and their complementary strands were
used to analyze mutants of ARE-2: M1,
5'-CTTGCAATCAAATCGTTCTAATTACAT-3'; M2,
5'-CTTGCAATCAAATCGTGCTCATTACAT-3'; M3,
5'-CTTGCAAGCACATCGTTCTAATTACAT-3'; M4,
5'-CTTGCAAGCACACTCGTGCTCATTACAT-3'; M5,
5'-CTTGCAAGCACACATCGTGCTCATTACAT-3'; M6,
5'-CTTGCAAGCACATCTGCTCATTACAT-3'; M7,
5'-CTTGCAAGCACATTGCTCATTACAT-3'.
The following oligonucleotides and their complementary strands were
used to analyze mutants of the SRE: SRE,
5'-ATGCATTGGGTACATCTTGTTCACATAGACA-3'; M1,
5'-ATGCATTGGTTAAATCTTGTTCACATAGACA-3'; M2,
5'-ATGCATTGGGTACATCTTTTTAACATAGACA-3'; M3,
5'-ATGCATTGGTTAAATCTTTTTAACATAGACA-3'; M4,
5'-ATGCATTGGGTACACATCTTGTTCACATAGACA-3'; M5,
5'-ATGCATTGGGTACACATCTTTGTTCACATAGACA-3'; M6,
5'-ATGCATTGGGTACACATCTTATGTTCACATAGACA-3'; M7,
5'-ATGCATTGGGTACATCTGTTCACATAGACA-3'; M8,
5'-ATGCATTGGGTACATTGTTCACATAGACA-3'; M9,
5'-ATGCATTGGGTACATCTTTGTTCACATAGACA-3'.
The -1,253 element found in the strongly androgen-dependent
CRISP-1 gene (50, 51, 52) and that differs only at position +7
from the consensus (7) was used as control SRE. Labeling
was carried out with DIG-11-dideoxy uridine triphosphate using the
terminal transferase (Roche Molecular Biochemicals). The
binding reaction was performed with 4 µl of in vitro
translated AR, 100 fmol of DIG-labeled probe, and 0.6 µg of
poly[d(I-C)] in 20 mM Tris, pH 7.9, 0.5
mM EDTA, 2.5 mM
MgCl2, 1.5 mM
dithiothreitol, 100 mg/ml Pefabloc SC, 15% glycerin, 0.1% NP-40, and
10-6 M R1881. The reaction
was incubated for 30 min at room temperature. For supershift
experiments, the monoclonal mouse antihuman AR antibody sc-7305X was
preincubated with in vitro translated AR for 15 min on ice
before adding the DIG-labeled DNA. For competition studies, a 25-fold
molar excess of unlabeled double-stranded oligonucleotide was added.
The complexes were separated on 5% polyacrylamide gels in 0.25x
Tris-borate-EDTA. The gels were blotted by a semidry procedure
onto positively charged nylon membranes (Roche Molecular Biochemicals) and developed by an anti-DIG antibody coupled to
alkaline phosphatase (Roche Molecular Biochemicals).
CSPD-Star (Roche Molecular Biochemicals) was used as
substrate for the alkaline phosphatase. The blots were exposed for 5
min to enhanced chemiluminescence (ECL) films (Amersham Pharmacia Biotech, Piscataway, NJ). Quantification was performed with the
Image Station (Kodak Digital Science, Rochester, NY).
Cell Culture, Preparation of Nuclear Extracts, and
Transfection
CV-1 cells were grown at 37 C in a 5% CO2
atmosphere in MEM, 10% FCS, 100 U/ml penicillin, 100 µg/ml
streptomycin, 4 mM L-glutamine. For extract
preparation, 1.5 x 106 cells seeded in
150-mm-diameter cell culture dishes were transfected with 15 µg of
pSG5/AR or empty plasmid and 30 µl of FuGene 6 reagent. After 5
h, the transfection medium was replaced by fresh culture medium with or
without 10-9 M R1881. After 24
h, the cells were trypsinized and centrifuged for 3 min at 1,500 rpm.
The cell pellet was washed twice with PBS, 2% FCS. Nuclear and
cytoplasmic proteins were extracted with the NE-PER kit (Pierce Chemical Co., Rockford, IL). Analysis of extracts was performed
by separating 20 µg of proteins on a 412% acrylamide gradient gel
(Novex system, Invitrogen) and transferring
them onto a polyvinylidene difluoride membrane using the
Novex transfer procedure. The AR was detected by the
sc-7305X anti-AR antibody (Santa Cruz Biotechnology, Inc.)
at a 1:1,000 dilution and the antimouse horseradish peroxidase antibody
at a 1:5,000 dilution. Detection was performed using the ECL kit and
ECL hyperfilms (Amersham Pharmacia Biotech). For the
transactivation assays, the cells were seeded in 96-well plates at a
concentration of 12,000 cells/100 µl/well in MEM supplemented as
described above except that 5% charcoal-stripped FCS was used. The
PC-3/AR cells were routinely cultured at 37 C in a 4.5%
CO2 atmosphere in RPMI 1640, 10% FCS, 100 U/ml
penicillin, 100 µg/ml streptomycin, 4 mM
L-glutamine, 600 µg/ml Geneticin. For the transactivation
assays, the cells were seeded at a concentration of 15,000 cells/100
µl/well in RPMI 1640 supplemented as described above except that 5%
charcoal-stripped FCS was used. For both cell lines, the transfection
was carried out 1819 h later using FuGene 6 in OPTI-MEM and 100 ng of
reporter plasmid. Expression plasmids for human AR, GR, or PR (100 ng
each) were cotransfected into CV-1 cells when indicated. Induction was
performed 5 h later by adding 10-9
M R1881, 10-9 M
dexamethasone, or 10-9 M R5020.
Measurement of luciferase activity was carried out 23 h later
after adding 100 µl of LucLite or LucLite Plus reagent (Packard
Instruments, Meriden, CT) in a Lumicount luminometer (Packard
Instruments). The activity of pGL3 promoter vector (Promega Corp.) was determined on parallel samples to assess transfection
efficiency. For all data points, the average value of six wells treated
in parallel was taken. The experiments were repeated at least three
times independently.
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. A. Cato, G. Langer, and J. Beekman for steroid
receptor cDNAs and Dr. T. Wirth for the pTATA plasmid. We are grateful
to Dr. A. Cato for the PC-3/AR cells. The expert technical assistance
of E. Wiecko, F. Knoth, and J. Wätzold was much appreciated.
 |
FOOTNOTES
|
---|
This work was supported in part by Grant 0310681B from the
Bundesministerium für Bildung und Forschung.
Abbreviations: ARE, Androgen response element; DIG,
digoxigenin; ECL, enhanced chemiluminescence; PB, probasin; Slp,
sex-limited protein; SRE, steroid response element; TK, thymidine
kinase.
Received for publication November 16, 2000.
Accepted for publication June 18, 2001.
 |
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