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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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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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). 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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, 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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).
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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). 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 5–7). No specific complex was
formed when using protein extracts from mock-transfected CV-1 cells
(lanes 2–4).
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). 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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). 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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), 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 (M4–M7).
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 (M1–M3). Any change in the spacing
brought about a complete loss of AR binding (M4–M9).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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-1–1253 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
manufacturer’s 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
manufacturer’s 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 4–12% 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 18–19 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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 S. M. Eacker, J. E. Shima, C. M. Connolly, M. Sharma, R. W. Holdcraft, M. D. Griswold, and R. E. Braun Transcriptional Profiling of Androgen Receptor (AR) Mutants Suggests Instructive and Permissive Roles of AR Signaling in Germ Cell Development Mol. Endocrinol., April 1, 2007; 21(4): 895 - 907. [Abstract] [Full Text] [PDF]
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 K. Schauwaers, K. De Gendt, P. T. K. Saunders, N. Atanassova, A. Haelens, L. Callewaert, U. Moehren, J. V. Swinnen, G. Verhoeven, G. Verrijdt, et al. Loss of androgen receptor binding to selective androgen response elements causes a reproductive phenotype in a knockin mouse model PNAS, March 20, 2007; 104(12): 4961 - 4966. [Abstract] [Full Text] [PDF]
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R.-S. Wang, S. Yeh, L.-M. Chen, H.-Y. Lin, C. Zhang, J. Ni, C.-C. Wu, P. A. di Sant'Agnese, K. L. deMesy-Bentley, C.-R. Tzeng, et al. Androgen Receptor in Sertoli Cell Is Essential for Germ Cell Nursery and Junctional Complex Formation in Mouse Testes Endocrinology, December 1, 2006; 147(12): 5624 - 5633. [Abstract] [Full Text] [PDF]
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P. Sipila, D. A. Pujianto, R. Shariatmadari, J. Nikkila, M. Lehtoranta, I. T. Huhtaniemi, and M. Poutanen Differential Endocrine Regulation of Genes Enriched in Initial Segment and Distal Caput of the Mouse Epididymis as Revealed by Genome-Wide Expression Profiling Biol Reprod, August 1, 2006; 75(2): 240 - 251. [Abstract] [Full Text] [PDF]
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E. Denolet, K. De Gendt, J. Allemeersch, K. Engelen, K. Marchal, P. Van Hummelen, K. A. L. Tan, R. M. Sharpe, P. T. K. Saunders, J. V. Swinnen, et al. The Effect of a Sertoli Cell-Selective Knockout of the Androgen Receptor on Testicular Gene Expression in Prepubertal Mice Mol. Endocrinol., February 1, 2006; 20(2): 321 - 334. [Abstract] [Full Text] [PDF]
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S. F. Sneddon, N. Walther, and P. T. K. Saunders Expression of Androgen and Estrogen Receptors in Sertoli Cells: Studies Using the Mouse SK11 Cell Line Endocrinology, December 1, 2005; 146(12): 5304 - 5312. [Abstract] [Full Text] [PDF]
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J. A. MacLean II, M. K. Rao, K. M.H. Doyle, J. S. Richards, and M. F. Wilkinson Regulation of the Rhox5 Homeobox Gene in Primary Granulosa Cells: Preovulatory Expression and Dependence on SP1/SP3 and GABP Biol Reprod, December 1, 2005; 73(6): 1126 - 1134. [Abstract] [Full Text] [PDF]
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 J. Brodie and I. J McEwan Intra-domain communication between the N-terminal and DNA-binding domains of the androgen receptor: modulation of androgen response element DNA binding J. Mol. Endocrinol., June 1, 2005; 34(3): 603 - 615. [Abstract] [Full Text] [PDF]
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K. Steketee, A. C J Ziel-van der Made, H. A G M van der Korput, A. B Houtsmuller, and J. Trapman A bioinformatics-based functional analysis shows that the specifically androgen-regulated gene SARG contains an active direct repeat androgen response element in the first intron J. Mol. Endocrinol., October 1, 2004; 33(2): 477 - 491. [Abstract] [Full Text] [PDF]
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P. I. Sadate-Ngatchou, D. J. Pouchnik, and M. D. Griswold Identification of Testosterone-Regulated Genes in Testes of Hypogonadal Mice Using Oligonucleotide Microarray Mol. Endocrinol., February 1, 2004; 18(2): 422 - 433. [Abstract] [Full Text] [PDF]
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M. I. Nonaka, Y. Hishikawa, N. Moriyama, T. Koji, R. T. Ogata, A. Kudo, H. Kawakami, and M. Nonaka Complement C4b-Binding Protein as a Novel Murine Epididymal Secretory Protein Biol Reprod, December 1, 2003; 69(6): 1931 - 1939. [Abstract] [Full Text] [PDF]
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C. Geserick, H.-A. Meyer, K. Barbulescu, and B. Haendler Differential Modulation of Androgen Receptor Action by Deoxyribonucleic Acid Response Elements Mol. Endocrinol., September 1, 2003; 17(9): 1738 - 1750. [Abstract] [Full Text] [PDF]
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 L. Callewaert, G. Verrijdt, V. Christiaens, A. Haelens, and F. Claessens Dual Function of an Amino-terminal Amphipatic Helix in Androgen Receptor-mediated Transactivation through Specific and Nonspecific Response Elements J. Biol. Chem., February 28, 2003; 278(10): 8212 - 8218. [Abstract] [Full Text] [PDF]
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M. K. Rao, C. M. Wayne, M. L. Meistrich, and M. F. Wilkinson Pem Homeobox Gene Promoter Sequences that Direct Transcription in a Sertoli Cell-Specific, Stage-Specific, and Androgen-Dependent Manner in the Testis in Vivo Mol. Endocrinol., February 1, 2003; 17(2): 223 - 233. [Abstract] [Full Text] [PDF]
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M. K. Rao, C. M. Wayne, and M. F. Wilkinson Pem Homeobox Gene Regulatory Sequences That Direct Androgen-dependent Developmentally Regulated Gene Expression in Different Subregions of the Epididymis J. Biol. Chem., December 6, 2002; 277(50): 48771 - 48778. [Abstract] [Full Text] [PDF]
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 M. R. Blackman, J. D. Sorkin, T. Munzer, M. F. Bellantoni, J. Busby-Whitehead, T. E. Stevens, J. Jayme, K. G. O'Connor, C. Christmas, J. D. Tobin, et al. Growth Hormone and Sex Steroid Administration in Healthy Aged Women and Men: A Randomized Controlled Trial JAMA, November 13, 2002; 288(18): 2282 - 2292. [Abstract] [Full Text] [PDF]
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G. Verrijdt, K. Schauwaers, A. Haelens, W. Rombauts, and F. Claessens Functional Interplay between Two Response Elements with Distinct Binding Characteristics Dictates Androgen Specificity of the Mouse Sex-limited Protein Enhancer J. Biol. Chem., September 13, 2002; 277(38): 35191 - 35201. [Abstract] [Full Text] [PDF]
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M. Fu, C. Wang, J. Wang, X. Zhang, T. Sakamaki, Y. G. Yeung, C. Chang, T. Hopp, S. A. W. Fuqua, E. Jaffray, et al. Androgen Receptor Acetylation Governs trans Activation and MEKK1-Induced Apoptosis without Affecting In Vitro Sumoylation and trans-Repression Function Mol. Cell. Biol., May 15, 2002; 22(10): 3373 - 3388. [Abstract] [Full Text] [PDF]
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