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Department of Molecular & Cellular Pharmacology (J.M.G., L.S.L., K.L.B.), University of Miami School of Medicine, Miami, Florida 33101; Geriatric Research, Education and Clinical Center (K.L.B.), Miami VA Medical Center, Miami, Florida 33125; Department of Human Genetics (D.M.R.), University of Michigan School of Medicine, Ann Arbor, Michigan 48109
Address all correspondence and requests for reprints to: Kerry L. Burnstein, Ph.D., Department of Molecular & Cellular Pharmacology (R-189), P.O. Box 016189, Miami, Florida 33101. E-mail: kburnste{at}miami.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) retained the capacity to
activate the internal responsive region, unlike a constitutively active
chimera harboring the GR amino-terminus and AR DNA binding domain
(GA). Thus, the AR amino terminus is the sole determinant for
androgen-specific regulation of the AR gene internal enhancer. These
findings support a model in which the amino termini of ARs bound to
HREs within the AR gene interact with an exclusive auxiliary factor(s)
to elicit androgen-specific regulation of AR messenger RNA. This is the
first example of androgen-specific response in which the necessary and
sufficient distinguishing capacity resides within the AR amino
terminus. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Subtle differences in individual HREs may underlie differential receptor DNA binding resulting in specific gene regulation in some cases. The prostatic gene probasin is regulated preferentially by AR in prostate cells. This regulation is exerted through two AREs in an upstream enhancer (17, 18) that, in vitro, reveal selective binding by the AR compared with the GR DBD (4, 5). Androgen-specific regulation of the secretory component gene also appears to derive from preferential AR binding to a distinct ARE in the promoter (6). Thus, specific androgen regulation of some genes may be explained by the capacity of the AR DBD to discriminate among HREs.
Steroid receptor function is strongly influenced by coactivator proteins, which interact directly with receptor and with the transcriptional machinery (reviewed in Refs. 19, 20, 21). A coactivator that interacts with one receptor in preference to others could provide a basis for specificity, but most coactivators identified thus far interact broadly with members of the nuclear receptor family. One coactivator, ARA 70, preferentially interacts with and increases the transcriptional activity of AR (22). However, this specificity of ARA 70 is not observed in all cell systems or assay conditions (23, 24, 25).
Heterotypic interactions with nonreceptor transcription factors may
contribute to androgen specificity (9, 10, 11, 12, 13, 14, 15, 26, 27, 28). Such
interactions represent an important component of tissue-specific gene
regulation and differential protein-protein interactions may form the
basis for steroid specificity of some target genes (9, 10, 11, 12, 13, 14, 15, 26, 27, 28, 29). For example, the transcription factor AML3/CBF
1
binds to a site adjacent to an HRE in the androgen-specific sex-limited
protein (Slp) gene enhancer and interacts better with AR than GR
in vitro (9). The amino termini of the
receptors may be involved in these interactions as this domain is
highly divergent even among closely related nuclear receptors. When the
AR amino terminus is fused to the GAL-4 DBD, transcriptional activity
is higher in a cell line in which HREs are preferentially regulated by
AR (29). However, an absolute requirement for the AR amino
terminus in androgen specificity has yet to be shown.
In addition to functions intrinsic to the three major receptor domains, intramolecular interactions are requisite for AR function. Interaction between the AR amino- and carboxyl-termini participates in ligand binding, gene transactivation, and regulation of AR stability (30, 31, 32, 33, 34). Furthermore, androgen-specific regulation of Slp is also dependent on AR domain interactions (16). Mapping of the AR domain(s) required for Slp enhancer specificity revealed a complex requirement for amino-carboxyl terminal interactions that promote cooperative binding of AR, but not GR, dimers to multiple HREs (16).
Although studies of androgen specificity have been limited to a small number of genes, several mechanisms appear to be used. In this report, we identify a new mechanism that operates in the androgen-specific regulation of AR messenger RNA (mRNA). An internal enhancer of the AR gene directs androgen-specific transcription, even when upstream of a heterologous reporter, in a cell-specific fashion (35, 36). Receptor chimeras comprised of amino-terminal, DBDs and ligand binding domains (LBDs) of AR and GR were used to define the domain requirements for androgen-specific regulation. We found that the information required for androgen-specific regulation of this unique enhancer resides solely in the AR amino terminus. Thus, the AR gene itself provides the long sought example for which steroid-specific regulation is determined by the highly divergent receptor amino terminus.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cells were transfected using the calcium phosphate coprecipitation
method as previously described (35). Supercoiled plasmid
DNA used for transfection was purified with QIAGEN columns
(Santa Clarita, CA). For reporter gene assays, all cells were cultured
in 60-mm plates and received 500 ng CMVhAR, RSVhGR, or the indicated
chimera, 10.0 µg reporter plasmid, and 1.0 µg 
-galactosidase
expression plasmid (CMV-gal) (to normalize for transfection
efficiency). Cells were incubated with the calcium-phosphate
precipitates for 5–7 h, glycerol shocked for 45 sec and then cultured
in McCoy’s media supplemented with 2% charcoal-stripped FBS (css
FBS). The indicated hormone [50 nM methyltrienolone
(R1881), 200 nM dexamethasone] or vehicle control was
added immediately after. For the experiments using constitutively
active receptors no hormone was added. Forty hours after hormone
treatment, transfected cells were harvested in 1x reporter lysis
buffer (Promega Corp., Madison, WI). -Galactosidase and
CAT activities from cell extracts were measured as previously described
(35, 36). The percent conversion of chloramphenicol to
acetylated forms was quantified by phosphorimage analysis using
ImageQuant software (Molecular Dynamics, Inc., Sunnyvale,
CA). Statistical analysis was performed using a Student’s t
test and SigmaPlot software (Jandel Scientific).
Reporter plasmids and expression vectors
Chimeric receptors containing combinations of mouse AR (mAR) or
rat GR (rGR) receptor domains were constructed as previously described
(16). The 
F581, R614, and R614H constructs were
obtained from Dr. L. Pinsky (37); the 538–614
construct was obtained from Dr. E. Wilson (38). The 350
wild-type (WT)-CAT reporter plasmid consists of exons D and E of the
human AR gene linked upstream from the tk promoter and CAT gene in the
vector pBLCAT2 and was described previously (35).
2XHRE-CAT (obtained from Dr. J. Cidlowski (NIEHS, Research Triangle
Park, NC) contains two tandem copies of an HRE (GRE) consensus sequence
linked to the minimal E1b promoter and CAT (39). The
I4-CAT reporter plasmid consists of exon D, intron 4 and exon E of the
human AR gene linked upstream from the tk promoter and CAT gene in the
vector pBLCAT2 (36) and was generated using an exonuclease
III digestion strategy as previously described (36).
The mI4-CAT reporter plasmid contains the 6.5-kb genomic fragment harboring mutations at ARE-1 and ARE-2. ARE-1 is changed from 5'-TGTCCT-3' to 5'-CGTCTT-3' and ARE-2 is changed from 5'-AGTACT-3' to 5'-AATATT-3' (altered base pairs are underlined) (36). The reporter plasmid was created using an exonuclease III digestion strategy similar to that described for I4-CAT (36) First, the AR genomic fragment encompassing intron 4 was amplified by PCR using human genomic DNA (Roche Molecular Biochemicals, Indianapolis, IN) as template and primers targeted to the 5' and 3' ends of intron 4. The primers also contained NotI sites as well as 10–12 bp of sequence complementary to the intron 4/exon D and exon E boundary within the AR gene. The forward primer was 5'-GTGGGCCAAGGCCTTGCCGCGTAAGGAAAAGGGAAGT-3'; the reverse primer was 5'-CCACGTGTAAGTTGCCGCGGCCCTGGAGAAGAAGAGG-3'. The 6.2-kb PCR product was gel purified and ethanol precipitated. Second, a NotI site was introduced into the intron/exon splice site of the WT 350-bp region or into a 350-bp region harboring mutations in both ARE-1 and ARE-2 (35), using the Clone Amp system (Life Technologies, Inc.). A first round of PCR was performed to generate the 5' and 3' halves of each mutated 350-bp region, respectively, (underlined nucleotides are mutated) using the mARE-1/mARE-2 mutant 350 bp construct as template (35). The primers for the first round of PCR were the forward (F) primer F-H3CAU (5'-CAUCAUCAUCAUGCCAGTGCCAAGCTTAAC-3') paired with the reverse (R) primer: R-350-NOT (5'-GTTGCGCGGCCGCGGCAAGGCCTTG-3') to produce the 5' fragment. The 3' fragments were generated using the reverse primer R-XbaCUA (5'-CUACUACUACUAGGATCCTCTAGACGGTAC-3') paired with the forward primer (underlined nucleotides are mutated): F-350NOT (5'-CCTTGCCGCGGCCGCGCAACTTACAC-3') to produce the 3' fragment. The resulting 5' and 3' fragments overlap approximately 10–15 bp surrounding the mutant sequences where they serve as template in the second round of PCR. The second round of PCR connects the 5' and 3' fragments using the F-H3CAU and R-XbaCUA primers. The resulting 350-bp fragments were cloned into the pAMP vector using uracil DNA glycosylase. The NotI-containing 350 bp regions were liberated by digestion with HindIII and XbaI and subcloned into a HindIII/XbaI digested pBLCAT2 vector for reporter gene assays. Each inserted 350-bp fragment was sequenced to confirm that each construct was in the 5'-3' orientation and upstream of the tk promoter of pBLCAT2 (40). The resulting constructs were called 350NotI-CAT and m350NotI-CAT. Each construct was linearized by digestion with NotI and gel purified. The intron fragment generated by PCR (described above) (50ng) and the NotI-digested 350NotI-CAT and m350NotI-CAT vectors were incubated with Exonuclease III (Stratagene, La Jolla, CA) for 45 sec. The total reaction volume was raised to 50 µl with TE buffer (pH 8.0), the ligated DNA phenol/chloroform extracted and ethanol precipitated and used for transformation of Epicurian Coli XL-10 Gold Ultracompetent cells (Stratagene, La Jolla, CA).
RNA isolation and analyses
Northern blot analyses and poly-A isolation were performed as
previously described (41, 42). RNase protection assays
were performed as previously described (35). Probe
fragments specifically protected by AR and GAPDH mRNA are 200 and 110
nucleotides, respectively.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether the AR gene is specifically regulated by androgen
in human osteosarcoma cells, we analyzed AR mRNA levels in U2OS cells
following androgen (R1881) or glucocorticoid (dexamethasone) treatment.
Northern blot analysis demonstrated that androgen treatment promoted a
2- to 2.5-fold increase in AR mRNA in U2OS cells (Fig. 1A
), as has been shown previously in
osteosarcoma cell lines (43, 44, 45). In contrast, RNase
protection assay showed that dexamethasone had no significant effect on
AR mRNA levels (Fig. 1B). RNase protection assay was used as this
method is more sensitive than Northern blotting. The failure of
glucocorticoid to regulate AR mRNA was not due to absence of functional
GR in this cell line because dexamethasone treatment led to induction
of the mouse mammary tumor virus-long terminal repeat (MMTV-LTR), which
responds to androgens and glucocorticoids in most cell lines (48, 49) (Fig. 1C). Androgen did not induce CAT activity from the
MMTV-LTR (Fig. 1C), consistent with lower levels of AR than GR in
osteosarcoma cells (50, 51, 52). The same results were
obtained using a reporter plasmid consisting of two copies of an HRE
linked to a minimal promoter and CAT (2XHRE-CAT) (data not shown).
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). This region was linked to
the heterologous tk promoter and CAT gene to assess androgen activation
in U2OS cells cotransfected with an AR complementary DNA (cDNA)
expression vector, CMVhAR. As shown previously (36),
the resulting reporter construct, I4-CAT, was induced by androgen (Fig. 2). Cotransfection with an AR cDNA was required as endogenous levels of
AR are low and not sufficient for activation of reporter genes (Fig. 1C
, 50). These data suggest that despite the location of this enhancer
downstream from the site of transcription initiation, this region
functions in androgen-mediated transcriptional regulation of AR
mRNA.
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). Consistent with this conclusion, a 350-bp fragment with only exons
D and E linked to tk-CAT (350WT-CAT) was regulated by androgen to the
same extent as I4-CAT (36 and Fig. 2, quantitation shown in Fig. 3B).
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). As a
control for GR function, glucocorticoid induction of the MMTV-CAT
reporter plasmid is shown (Fig. 3A). Quantitation of androgen
vs. glucocorticoid regulation of the 350WT-CAT and I4-CAT
constructs is shown in Fig. 3B. The failure of GR to regulate either
350WT-CAT or I4-CAT is unlikely to be due to insufficient GR levels as
a robust induction of MMTV-CAT was observed in dexamethasone treated
U2OS cells expressing the GR cDNA (Fig. 3A). This induction is
comparable with that observed in androgen-treated cells expressing
CMVhAR (Fig. 4, WT). These results
indicate that the HRE-containing AR gene regions retain the
androgen specificity exhibited by the native AR gene.
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F581 and R614 (37), lack Phe at position
581 and Arg at position 614, respectively. These amino acids, located
just C-terminal to the first zinc finger (F581) and second zinc
finger (R614) of AR, are highly conserved among steroid receptors
and are required for AR interaction with AREs (37). A
third DNA binding mutant, R614H, harbors an arginine to histidine
substitution at position 614 (37). 538–614 lacks the
region from position 538 to 614, which includes both zinc fingers
(38). Each mutant receptor has been shown to be expressed
in heterologous cells (37, 38, 54) and to bind androgen
(37, 38, 54). Based on the ability of the F581,
R614, and R614H mutant receptors to bind ligand (37),
these receptors are expected to be predominantly nuclear in the
presence of androgen. However, localization studies of these mutants
have not been reported. The 538–614 receptor has been shown to
translocate to the nucleus in the presence of androgen, albeit to a
lesser extent than WT AR (38). Consistent with reports
from other investigators (37, 54), we found that these
mutant receptors did not transactivate reporter gene activity from the
MMTV-LTR (Fig. 4). Similarly, the mutant receptors did not
transactivate 350WT-CAT (Fig. 4). These data, in conjunction with our
identification of HREs within the 350-bp and 6.5-kb fragments whose
mutation abrogates induction (35 and Fig. 2), indicate that AR binding
to HREs in the internal enhancer is essential for androgen regulation
of this region.
The amino terminus of AR is required for transactivation of the
internal enhancer of the AR gene
To define AR domains responsible for the androgenspecific
regulation of the AR gene internal enhancer, we used a panel of AR/GR
chimeric receptors (Fig. 5A). In these
chimeras, the amino terminal region, DBD and LBD originate from either
the mouse AR or the rat GR cDNAs (16). Chimeric receptors
are named by the origin of each domain with A designating AR and G,
indicating GR. For example, a chimeric receptor containing an AR amino
terminus, a GR DBD and a GR LBD is designated AGG. Expression of the
mouse AR, rat GR and all chimeric receptors is under the control of the
CMV promoter (16).
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). In contrast, none of the
chimeric receptors containing a GR amino-terminus induced 350WT-CAT
(Fig. 5B). Consistent with earlier findings (16), all the
chimeric receptors transactivated a simple, nonspecific enhancer
comprised of two HREs linked to a minimal promoter and CAT (2xHRE-CAT)
(Fig. 5C). The AGA chimera evoked a more modest induction of 350WT-CAT
compared with the control (AAA) and chimeric receptors, AGG and AAG.
However, the AGA chimera was also a poor transactivator of MMTV-CAT
relative to the other receptors (Fig. 5D). Thus, this chimeric receptor
may be inherently weaker at more complex regulatory regions such as the
MMTV-LTR, which consists of multiple HREs as well as other
transcription factor binding sites (48, 49).
To determine whether androgen-specific regulation of the genomic 6.5-kb
internal enhancer also required the AR amino terminus, AGG and GAA were
tested in parallel with both I4-CAT and 350WT-CAT. These chimeric
receptors displayed the same specificity for I4-CAT as for 350WT-CAT;
AGG but not GAA transactivated the AR gene internal enhancer (Fig. 6). Thus, androgen specificity is
retained even though the spatial organization of HREs differs
dramatically between the 6.5-kb and 350-bp enhancer regions.
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and 6B), AGA had little
effect on mARE1,2-CAT (Fig. 6B). Because only the DBD portion of AGA is
from GR, these data suggest that the GR DBD can bind to HREs within the
AR gene enhancer.
A ligand binding domain is not required for regulation of the AR
gene internal enhancer
The experiments discussed above show that in the context of a
full-length receptor the AR amino terminus is the primary determinant
of androgen-specific regulation of the AR gene internal enhancer. To
determine whether a receptor LBD is required for regulation of
350WT-CAT, constitutively active receptors lacking the LBD were
employed in reporter gene assays. These constructs contain either the
AR amino-terminus and an AR DBD (AA) or the GR amino-terminus and an
AR DBD (GA). Both AA and GA induced reporter gene activity
from the nonspecific 2XHRE-CAT and MMTV-CAT reporter constructs
normalized to the parent vector CMV5 (Fig. 7A). In contrast AA, but not GA,
activated the 350-bp region (Fig. 7B). Thus, the AR amino terminal
domain is necessary for specificity and sufficient when coupled to a
receptor DBD, even in the absence of an LBD.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We examined regulation of a reporter plasmid containing the 350-bp AR
gene enhancer fragment, which contains exon D and E sequences linked
upstream of the tk promoter and CAT gene. A panel of chimeric receptors
composed of AR and GR domains was used to identify the region of the
AR required for the androgen specificity exhibited by this AR gene
enhancer. Only receptors containing an AR amino terminal segment
(parental AAA and chimeras AGA, AGG, AAG) transactivated this enhancer
(Fig. 5B). Similar results were obtained with the 6.5-kb genomic
enhancer, which consists of exons D and E separated by intron 4,
suggesting that no critical additional regulatory sequences reside in
the intron and that specificity of enhancer regulation occurs when the
HREs are in the context of the native enhancer. The importance of the
AR amino terminus in regulating the AR gene enhancer was verified
further by experiments showing that of the two truncated,
constitutively active receptors tested (AA and GA), only AA
activated the internal AR gene enhancer (Fig. 7B). This result not only
confirms the requirement for the AR amino terminus but shows that the
AR LBD is not specifically involved in conferring androgen selective
regulation of the AR gene enhancer.
Similar to other nuclear receptors, AR contains an AF-1 (activation function 1) region in the amino terminal domain, which participates in gene transactivation. The AF-1 regions of nuclear receptors are highly variable in both sequence and length and their mode of action is still incompletely understood. One general mechanism by which the AF-1 region has been proposed to function is through recruitment of basal transcription factors and RNA polymerase to target gene promoters (55). More recent data supports indirect interaction between nuclear receptors and the transcriptional machinery via coactivators (19, 20, 21). The amino terminal AF-1 of AR contacts the p160 family of coregulators, which includes SRC-1 (25, 56, 57), and this interaction is mechanistically more important for transactivation by AR than the ligand-dependent interaction between SRC-1 and the AF-2 region of the AR LBD (25). By contrast, ligand-dependent AF-2 interaction with coactivators appears to be more critical for transactivation by other nuclear receptors including ER (19, 20, 21). A new mode of coactivator recruitment by AR has been proposed (33, 58, 59) that depends on the well established AR amino-carboxyl terminal interaction (30, 31, 32, 33, 34). The coactivators CBP and F-SRC-1 enhance the amino-carboxyl terminal interaction of the rat AR suggesting that coactivators may function in this manner (33). The human AR amino terminus contains WXXLF and FXXLF sequences that strongly interact with AF-2 (59). Unlike other nuclear receptors in which coactivators interact with receptor AF-2 regions via LXXLL sequences, SRC1 and TIF2 bind AR amino terminal and DBD regions in an LXXLL-independent manner (58, 59). Thus, the AR amino terminal WXXLF and FXXLF sequences may compete with coactivators for binding to the AR AF-2 region (58, 59). Despite these differences in the mechanisms of coactivator function for AR compared with other nuclear receptors, p160 coactivators are unlikely to confer androgen specificity as these factors interact widely with and enhance activity of numerous members of the nuclear receptor superfamily (19, 20, 21).
The AR amino terminus is unique in that two different polymorphic trinucleotide repeat regions encoding polyglutamine and polyglycine tracts are present. ARs with shorter or longer than average polyglutamine repeats have been associated with altered transcriptional activity (60, 61) and receptor levels (62). These repeats may contribute to androgen-specific gene responses by directing exclusive interaction with other factors. In addition, the AR amino terminus was shown to contain a small region that is involved in interaction with the retinoblastoma protein (Rb) (63, 64). Although Rb enhances both AR and GR transactivation, AR but not GR function appears to depend on Rb (63). Because AR function is diminished in the absence of Rb, differences in Rb status are unlikely to underlie androgen specificity.
Androgen regulation of the AR gene internal enhancer regions was demonstrated with both human and mouse AR. The amino acid sequences of the mouse and human AR N termini are about 90% sequence identical (65), with largely conservative changes. Perhaps the most striking differences are in placement and length of the two polyamino acid tracts (65). The polyglutamine tract is about 100 amino acids closer to the carboxyl terminus in mouse and is disrupted by histidines (65). The polyglycine tract, located near the DBD, contains about twenty glycines in human but only four or five in mouse (65). A physiological correlate to these species differences is not known, and distinct differences (for example in transcriptional activity) between mAR and hAR in vitro have not been noted.
We have found that the carboxyl terminus of GR can substitute for that
of AR because the full-length receptor chimeras, AGG and AAG, regulate
the AR gene internal enhancer. Furthermore, the receptor, AA,
lacking a carboxyl terminal region also activates the AR gene enhancer,
suggesting that the amino-carboxyl interaction of AR is not essential
for androgen-specific regulation of this region in reporter gene
assays. However, the AR amino-carboxyl interaction may be important in
androgen-specific regulation of native genes. The activity of AA
also suggests that neither the AR nor GR carboxyl terminal regions are
needed to maintain the amino terminal domain of AR in an appropriate
conformation for protein-protein contacts.
Androgen-specific regulation of the AR gene enhancer occurs through a
mechanism that contrasts with the other modes of androgen specificity
described for androgen target genes like probasin, secretory component
and Slp. Further, unlike many other androgen-specific enhancers, which
GR can activate in certain cell lines, the AR gene enhancer is not
regulated by GR in any cellular context investigated thus far (data not
shown). One mechanism, used by the probasin gene, involves differential
binding affinities of the AR and GR DBDs (5). Although not
measured directly, the GR DBD appears to bind effectively to the AR
gene AREs as chimeras containing the GR DBD (AGA, AGG) transactivate
both the 350-bp and 6.5-kb enhancers (Fig. 6). Thus, androgen
specificity of the AR gene is not exerted through differential DNA
binding. The Slp gene is also regulated in an androgen-specific
fashion. However unlike probasin, specificity derives from a complex
interplay of interactions that occur between the AR domains as well as
between receptor and other transcription factors, notably AML3/CBF1
(9, 10, 16). The AR amino terminus is not sufficient for
androgen- specific regulation of Slp as specificity depends in part
on the ability of the AR DBD, but not the GR DBD, to evade suppressive
effects exerted by neighboring factors (16). Further,
interaction between AR’s amino and carboxyl termini underlies
cooperative DNA binding by AR dimers (16). In contrast to
Slp, androgen regulation of the AR gene enhancer occurs through an
apparently simpler mechanism requiring only the AR amino terminus for
specificity.
In summary, our results indicate that the AR amino terminus plays a dominant role in androgen-specific regulation of an androgen target gene. This finding supports longstanding theories that steroid-specific regulation of target gene enhancers occurs via distinct and specific associations between nonreceptor auxiliary factors and the highly divergent amino terminal regions of receptors. For the androgenspecific regulation described in this study, a putative intermediary factor appears to be selective for the AR amino terminus, as the GR amino terminus cannot substitute. This factor may be a previously described DNA binding protein or a coactivator that interacts in this case to confer stringent specificity not previously demonstrated. Although the contribution from nonreceptor transcription factors in steroid regulated gene expression is clear, no model systems have been elucidated in which the specific and perhaps broad role of receptor amino termini in maintaining steroid specific responses could be assessed. Our finding that the AR amino terminus is the sole contributor to androgen-specific regulation of the AR gene enhancer now provides the means to identify which factors impart this specificity.
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Acknowledgments |
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Footnotes |
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2 Received support from the American Heart Association-Florida
Affiliate (Fellowship No. 9604012) and the NIH (T-32-HL-07188).
Received August 10, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 is required
for androge-specific activation of the enhancer of the mouse
sex-limited protein (Slp) gene. J Biol Chem 274:30624–30630 isoform. Mol Endocrinol 13:117–128This article has been cited by other articles:
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5 µg) was analyzed by Northern blotting using AR and
GAPDH cDNA probes. B, Total RNA (20 µg) was analyzed by ribonuclease
protection assay. GAPDH mRNA was assessed in each experiment to control
for loading. C, U2OS cells were cotransfected with MMTV-CAT and a
CMV
