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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {alpha}-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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go). 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).



View larger version (38K):
[in this window]
[in a new window]
 
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.

 
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. 1Go). 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. 1Go).

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. 1Go), 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. 2Go). 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.



View larger version (34K):
[in this window]
[in a new window]
 
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.

 
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. 3AGo, 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. 3BGo, 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. 8Go).



View larger version (43K):
[in this window]
[in a new window]
 
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.

 


View larger version (29K):
[in this window]
[in a new window]
 
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.

 
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. 4AGo). 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.



View larger version (29K):
[in this window]
[in a new window]
 
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.

 
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. 4BGo). 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. 5Go, lanes 5–7). No specific complex was formed when using protein extracts from mock-transfected CV-1 cells (lanes 2–4).



View larger version (49K):
[in this window]
[in a new window]
 
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.

 
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. 6AGo). 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.



View larger version (49K):
[in this window]
[in a new window]
 
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.

 
The steroid selectivity of Pem ARE-2 was investigated by performing similar transactivation experiments in CV-1 cells (Fig. 6BGo). 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. 6BGo). 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. 6BGo).

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 1Go), 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. 7Go, A and B), and the amount of complex formed was quantified (Fig. 7CGo). 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.


View this table:
[in this window]
[in a new window]
 
Table 1. DNA Sequence Comparison of AREs

 


View larger version (45K):
[in this window]
[in a new window]
 
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.

 
Next, we examined ARE-2. Similar changes were introduced into this element (Fig. 8AGo), and binding by the AR was analyzed as described above with the EMSA (Fig. 8BGo) and quantified (Fig. 8CGo). 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. 9Go). 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).



View larger version (34K):
[in this window]
[in a new window]
 
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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 1Go). 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. 9Go), 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. 2Go).

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 1Go). 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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-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.


    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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Lucas PC, Granner DK 1992 Hormone response domains in gene transcription. Annu Rev Biochem 61:1131–1173[CrossRef][Medline]
  2. Tsai MJ, O’Malley BW 1994 Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 63:451–486[CrossRef][Medline]
  3. Kokontis JM, Liao S 1999 Molecular action of androgen in the normal and neoplastic prostate. Vitam Horm 55:219–307[Medline]
  4. Freedman LP 1992 Anatomy of the steroid receptor zinc finger region. Endocr Rev 13:129–145[Medline]
  5. Luisi BF, Xu WX, Otwinowski Z, Freedman LP, Yamamoto KR, Sigler PB 1991 Crystallographic analysis of the interaction of the glucocorticoid receptor with DNA. Nature 352:497–505[CrossRef][Medline]
  6. Zilliacus J, Wright APH, Carlstedt-Duke J, Gustafsson JA 1995 Structural determinants of DNA-binding specificity by steroid receptors. Mol Endocrinol 9:389–400[Medline]
  7. Roche PJ, Hoare SA, Parker MG 1992 A consensus DNA-binding site for the androgen receptor. Mol Endocrinol 6:2229–2235[Abstract]
  8. Nelson CC, Hendy SC, Shukin RJ, et al. 1999 Determinants of DNA sequence specificity of the androgen, progesterone, and glucocorticoid receptors: evidence for differential steroid receptor response elements. Mol Endocrinol 13:2090–2107[Abstract/Free Full Text]
  9. Zhou Z, Corden JL, Brown TR 1997 Identification and characterization of a novel androgen response element composed of a direct repeat. J Biol Chem 272:8227–8235[Abstract/Free Full Text]
  10. Verrijdt G, Schoenmakers E, Haelens A, et al. 2000 Change of specificity mutations in androgen-selective enhancers: evidence for a role of differential DNA binding by the androgen receptor. J Biol Chem 275:12298–12305[Abstract/Free Full Text]
  11. Chang C, Saltzman A, Yeh S, et al. 1995 Androgen receptor: an overview. Crit Rev Eukaryot Gene Expr 5:97–125[Medline]
  12. Adler AJ, Scheller A, Robins DM 1993 The stringency and magnitude of androgen-specific gene activation are combinatorial functions of receptor and nonreceptor binding site sequences. Mol Cell Biol 13:6326–6335[Abstract]
  13. Scarlett CO, Robins DM 1995 In vivo footprinting of an androgen-dependent enhancer reveals an accessory element integral to hormone response. Mol Endocrinol 9:413–423[Abstract]
  14. Kasper S, Rennie PS, Bruchovsky N, et al. 1994 Cooperative binding of androgen receptors to two DNA sequences is required for androgen induction of the probasin gene. J Biol Chem 269:31763–31769[Abstract/Free Full Text]
  15. Cleutjens KBJM, van Eekelen CCJM, van der Korput HAGM, Brinkmann AO, Trapman J 1996 Two androgen response regions cooperate in steroid hormone regulated activity of the prostate-specific antigen promoter. J Biol Chem 271:6379–6388[Abstract/Free Full Text]
  16. Reid KJ, Hendy SC, Saito J, Sorensen P, Nelson CC 2001 Two classes of androgen receptor elements mediate cooperativity through allosteric interactions. J Biol Chem 276:2943–2952[Abstract/Free Full Text]
  17. Strähle U, Boshart M, Klock G, Stewart F, Schütz G 1989 Glucocorticoid- and progesterone-specific effects are determined by differential expression of the respective hormone receptors. Nature 339:629–632[CrossRef][Medline]
  18. Rundlett SE, Miesfeld RL 1995 Quantitative differences in androgen and glucocorticoid receptor DNA binding properties contribute to receptor-selective transcriptional regulation. Mol Cell Endocrinol 109:1–10[CrossRef][Medline]
  19. List HJ, Lozano C, Lu J, Danielsen M, Wellstein A, Riegel AT 1999 Comparison of chromatin remodeling and transcriptional activation of the mouse mammary tumor virus promoter by the androgen and glucocorticoid receptor. Exp Cell Res 250:414–422[CrossRef][Medline]
  20. Celis L, Claessens F, Peeters B, Heyns W, Verhoeven G, Rombauts W 1993 Proteins interacting with an androgen-responsive unit in the C3(1) gene intron. Mol Cell Endocrinol 94:165–172[CrossRef][Medline]
  21. Lu S, Jenster G, Epner DE 2000 Androgen induction of cyclin-dependent kinase inhibitor p21 gene: role of androgen receptor and transcription factor Sp1 complex. Mol Endocrinol 14:753–760[Abstract/Free Full Text]
  22. Yeh S, Chang C 1996 Cloning and characterization of a specific coactivator, ARA70, for the androgen receptor in human prostate cells. Proc Natl Acad Sci USA 93:5517–5521[Abstract/Free Full Text]
  23. Müller JM, Isele U, Metzger E, et al. 2000 FHL2, a novel tissue-specific coactivator of the androgen receptor. EMBO J 19:359–369[Abstract/Free Full Text]
  24. McKenna NJ, Xu J, Nawaz Z, Tsai SY, Tsai MJ, O’Malley BW 1999 Nuclear receptor coactivators: multiple enzymes, multiple complexes, multiple functions. J Steroid Biochem Mol Biol 69:3–12[CrossRef][Medline]
  25. Edwards DP 1999 Coregulatory proteins in nuclear hormone receptor action. Vitam Horm 55:165–218[Medline]
  26. Scheller A, Hugues E, Golden KL, Robins DM 1998 Multiple receptor domains interact to permit, or restrict, androgen-specific gene activation. J Biol Chem 273:24216–24222[Abstract/Free Full Text]
  27. Nelson SA, Robins DM 1997 Two distinct mechanisms elicit androgen-dependent expression of the mouse sex-limited protein gene. Mol Endocrinol 11:460–469[Abstract/Free Full Text]
  28. Ho KC, Marschke KB, Tan J, Power SGA, Wilson EM, French FS 1993 A complex response element in intron 1 of the androgen-regulated 20 kDa protein gene displays cell type-dependent androgen receptor specificity. J Biol Chem 268:27226–27235[Abstract/Free Full Text]
  29. Claessens F, Alen P, Devos A, Peeters B, Verhoeven G, Rombauts W 1996 The androgen-specific probasin response element 2 interacts differentially with androgen and glucocorticoid receptors. J Biol Chem 271:19013–19016[Abstract/Free Full Text]
  30. Verrijdt G, Schoenmakers E, Alen P, et al. 1999 Androgen specificity of a response unit upstream of the human secretory component gene is mediated by differential receptor binding to an essential androgen response element. Mol Endocrinol 13:1558–1570[Abstract/Free Full Text]
  31. Schoenmakers E, Alen P, Verrijdt G, et al. 1999 Differential DNA binding by the androgen and glucocorticoid receptors involves the second Zn-finger and a C-terminal extension of the DNA-binding domains. Biochem J 341:515–521[CrossRef][Medline]
  32. Schoenmakers E, Verrijdt G, Peeters B, Verhoeven G, Rombauts W, Claessens F 2000 Differences in DNA binding characteristics of the androgen and glucocorticoid receptors can determine hormone-specific responses. J Biol Chem 275:12290–12297[Abstract/Free Full Text]
  33. Aumais JP, Lee HS, DeGannes C, Horsford J, White JH 1996 Function of directly repeated half-sites as response elements for steroid hormone receptors. J Biol Chem 271:12568–12577[Abstract/Free Full Text]
  34. Maiti S, Doskow J, Li S, Nhim RP, Lindsey JS, Wilkinson MF 1996 The Pem homeobox gene: androgen-dependent and -independent promoters and tissue-specific alternative RNA splicing. J Biol Chem 271:17536–17546[Abstract/Free Full Text]
  35. Lindsey JS, Wilkinson MF 1996 Pem: a testosterone- and LH-regulated homeobox gene expressed in mouse Sertoli cells and epididymis. Dev Biol 179:471–484[CrossRef][Medline]
  36. Sutton KA, Maiti S, Tribley WA, et al. 1998 Androgen regulation of the Pem homeodomain gene in mice and rat Sertoli and epididymal cells. J Androl 19:21–30[Abstract/Free Full Text]
  37. Maiti S, Meistrich ML, Wilson G, et al. 2001 Irradiation selectively inhibits expression from the androgen-dependent Pem homeobox gene promoter in Sertoli cells. Endocrinology 142:1567–1577[Abstract/Free Full Text]
  38. Smale ST 1997 Transcriptional initiation from TATA-less promoters within eukaryotic protein-coding genes. Biochim Biophys Acta 1351:73–88[Medline]
  39. Heinemeyer T, Wingender E, Reuter I, et al. 1998 Databases on transcriptional regulation: TRANSFAC, TRRD, and COMPEL. Nucleic Acids Res 26:362–367[Abstract/Free Full Text]
  40. Couette B, Le Ricousse S, Fortin D, Rafestin-Oblin ME, Richard-Foy H 1994 The establishment of the long terminal repeat of the mouse mammary tumor virus into CV-1 cells allows a functional analysis of steroid receptors. Biochim Biophys Acta 1219:607–612[Medline]
  41. Szapary D, Xu M, Simon Jr SS 1996 Induction properties of a transiently transfected glucocorticoid-responsive gene vary with glucocorticoid receptor concentration. J Biol Chem 271:30576–30582[Abstract/Free Full Text]
  42. Quarmby VE, Yarbrough WG, Lubahn DN, French FS, Wilson EM 1990 Autologous down-regulation of androgen receptor messenger ribonucleic acid. Mol Endocrinol 4:22–28[Abstract]
  43. Shan LX, Rodriguez MC, Jänne OA 1990 Regulation of androgen receptor protein and mRNA concentrations by androgens in rat ventral prostate and seminal vesicles and in human hepatoma cells. Mol Endocrinol 4:1636–1646[Abstract]
  44. Wiren KM, Zhang X, Chang C, Keenan E, Orwoll ES 1997 Transcriptional up-regulation of the human androgen receptor by androgens in bone cells. Endocrinology 138:2291–2300[Abstract/Free Full Text]
  45. Marschke KB, Tan J, Kupfer SR, Wilson EM, French FS 1995 Specificity of simple hormone response elements in androgen regulated genes. Endocrine 3:819–825
  46. Dahlman-Wright K, Siltala-Roos H, Carlstedt-Duke J, Gustafsson JA 1990 Protein-protein interactions facilitate DNA binding by the glucocorticoid receptor DNA-binding domain. J Biol Chem 265:14030–14035[Abstract/Free Full Text]
  47. Umesono KK, Murakami KK, Thompson CC, Evans RM 1991 Direct repeats as selective response elements for the thyroid receptor, retinoic acid, and vitamin D3 receptors. Cell 65:1255–1266[Medline]
  48. Devereux J, Haeberli P, Smithies O 1984 A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387–395[Abstract]
  49. Annweiler A, Zwilling S, Hipskind RA, Wirth T 1993 Analysis of transcriptional stimulation by recombinant Oct proteins in a cell-free system. J Biol Chem 268:2525–2534[Abstract/Free Full Text]
  50. Schwidetzky U, Schleuning WD, Haendler B 1997 Isolation and characterization of the androgen-dependent mouse cysteine-rich secretory protein-1 (CRISP-1) gene. Biochem J 321:325–332[Medline]
  51. Haendler B, Habenicht UF, Schwidetzky U, Schüttke I, Schleuning WD 1997 Differential androgen regulation of the murine genes for cysteine-rich secretory proteins (CRISP). Eur J Biochem 250:440–446[Abstract]
  52. Haendler B, Schüttke I, Schleuning WD 2001 Androgen receptor signalling: comparative analysis of androgen response elements and implication of heat-shock protein 90 and 14-3-3{eta}. Mol Cell Endocrinol 173:63–73[CrossRef][Medline]