©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Androgen Receptor Is Transcriptionally Suppressed by Proteins That Bind Single-stranded DNA (*)

Michael E. Grossmann , Donald J. Tindall (§)

From the (1) Departments of Urology and Biochemistry/Molecular Biology, Mayo Foundation, Rochester, Minnesota 55905

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The androgen receptor (AR) is a nuclear transcription factor that is essential for development of the male urogenital tract. In the current work, we have characterized the mouse androgen receptor suppressor (mARS). A single, 20-base pair, region (TCCCCCCACCCACCCCCCCT) was sufficient for suppression in chloramphenicol acetyltransferase assays. Northern analysis indicated that translational regulation is not necessary for the suppression. Analysis of the AR mRNA half-life indicated that the mARS does not affect AR RNA degradation. Gel mobility assays showed that the mARS is bound by multiple proteins that can recognize single-stranded DNA and RNA. In addition, differing proteins are expressed in distinct tissues. Purification of some of these proteins has shown that a doublet of 33 and 35 kDa binds to the G-rich strand and that a 52-kDa protein binds to the C-rich strand. Southwestern blots have confirmed that these proteins are indeed recognized by the mARS. The results of these experiments indicate that the AR 5`-untranslated region contains a suppressor element that can be bound by multiple proteins. The mARS appears to be acting either by altering transcription initiation or blocking transcription elongation. Characterization of this suppressor may provide insight into the physiological means by which the AR is regulated.


INTRODUCTION

The androgen receptor (AR)() is a nuclear phosphoprotein that serves as a transcription factor whose function is modulated by testosterone and dihydrotestosterone. The AR is essential during development and maintenance of the male urogenital organs and has been implicated in a number of diseases. It is expressed at a relatively low level and has been shown to be regulated in a very complex manner that is dependent on both tissue and developmental effects (1) . Recent studies have shown that the AR can be regulated by androgens (2, 3) , follicle-stimulating hormone (4) , epidermal growth factor (5) , and the cAMP pathway (6, 7) . Tissue-specific factors regulating the AR have not yet been defined, although an age-dependent factor has been implicated in the expression of AR during development of the rat liver (8) . The regulation of the AR occurs at several levels, including translation, mRNA stability (9) , and transcription (10) . Further definition of the specific factors involved in AR regulation should allow a better understanding of how the AR is controlled during normal and diseased states.

The AR cDNA has been cloned and characterized from several different species (11, 12, 13, 14) . In addition, the 5`-flanking region of the gene has been cloned and shown to contain two promoters. Promoter-1 is the 5`-most promoter and is located approximately 1000 bp upstream from the ATG translation start site. For the purposes of this paper, the 5`-most site of transcription initiation from promoter-1 of the mouse AR will be numbered +1 (13) . Promoter-2 is located approximately 160 bp downstream from promoter-1 (15) . The function(s) of the large region from promoter-2 to the translation start site remained unknown until a novel regulatory element in this region was discovered by this laboratory (16) . It was shown using CAT assays that the 5`-untranslated region of the AR contains a cis-acting suppressor element that was termed the mouse androgen receptor suppressor (mARS). The mARS is capable of down-regulating AR or thymidine kinase promoters if it is inserted 3` to the site of transcription initiation. It was also shown that the mARS is capable of binding to proteins using both footprinting and bandshift assays. In the current work, we further characterize the specific sequences of the mARS, the mechanism of mARS action, and the proteins that bind to the mARS.


EXPERIMENTAL PROCEDURES

Construction of CAT Expression Plasmids

mARS mutants were constructed by annealing oligonucleotides that contained SalI sites and ligating the double-stranded nucleotides into the XhoI site in the thymidine kinase promoter containing pBLCAT2 vector. Clones were sequenced using the fmol sequencing system (Promega).

Cell Culture and Transfection Procedure

GT1-7 cells are a mouse hypothalamic cell line (kindly provided by Dr. P. Mellon see Ref. 17) and express low levels of AR transcripts as shown by analysis of steady-state levels of RNA on a Northern blot (data not shown). The cells were grown to confluency in T-175 flasks using Dulbecco's modified Eagle's medium (with pen/strep) with 5% charcoal-stripped fetal calf serum, 5% charcoal-stripped horse serum. The cells were harvested using trypsin-EDTA, washed (2 times), and counted. Cells (20 10) were pelleted, resuspended in 400 µl of 0.1% glucose/phosphate-buffered saline containing 15 µg of CAT vector and 5 µg of RSV--GAL vector, and electroporated at 960 microfarads and 0.35 kV. The cells were incubated on ice for 7 min, resuspended in 1 ml of phosphate-buffered saline with 4% charcoal-stripped fetal calf serum, incubated at room temperature for 7 min, and resuspended in 42 ml of Dulbecco's modified Eagle's medium with 5% charcoal-stripped fetal calf serum, 5% charcoal-stripped horse serum. Ten ml of cell suspension were plated into each of four 100 mM culture dishes and harvested 48 h later with phosphate-buffered saline, 2 mM EDTA. Cells were lysed in 100 mM Tris (pH 7.8), 0.1% Triton X-100, and the resulting supernatant was assayed for CAT and RSV--GAL activity. In brief, the CAT assay consisted of 50 µl of nuclear extract and 100 µl of Tris/Triton X-100 buffer placed in a scintillation vial and incubated at 70 °C for 10 min. The mixture was cooled to room temperature and overlaid with 100 µl of acetyl-CoA mixture (2.5 mM chloramphenicol, 0.1 M Tris (pH 7.8), 2 µl of [H]acetyl-CoA (5 Ci/mM)) added, and 2 ml of Ecoscint O scintillation fluid. After acetylation, the H entered the organic phase and was counted a minimum of 5 times over 2-6 h. CAT activity was calculated by linear regression and normalized to RSV--GAL values. Cells that were used in the RNA half-life experiments were treated with actinomycin D at a final concentration of 5 µg/ml and harvested 48 h after transfection.

RNA Preparations

Total RNA was isolated from tissues or cells by lysing the cells with 3 M LiCl, 6 M urea. The samples were homogenized on ice for 1 min with a Polytron and incubated on ice for 3-18 h. The samples were centrifuged 20 min at 25,000 rpm. The pellet was resuspended in ES (0.1% SDS, 0.2 mM EDTA), extracted with phenol once and chloroform/isoamyl alcohol twice, and precipitated by adding one-tenth volume of 3 M NaAc/HAc (pH 5.2) and 2.5 volumes of EtOH. The RNA pellet was resuspended in water and quantitated. RNA prepared from transfected cells was treated with 0.3 units of DNase I/20 µg of RNA for 10 min at 30 °C.

RNA Separation and Blotting

RNA was separated by electrophoresis on a 1% agarose gel that contained 2% formaldehyde using 1 MOPS buffer (0.02 M MOPS, 0.005 M NaAc, 0.001 M EDTA). The RNA was transferred to a Hybond-N membrane (Amersham Corp.) and UV cross-linked to the membrane using a Stratalinker (Stratagene). The probes were labeled by denaturing 125 ng of DNA in 12 µl of water by heating to 100 °C for 3 min and adding 4.5 µl of oligonucleotide labeling buffer (0.2 mM dCTP, 0.2 mM dTTP, 0.2 mM dGTP, 50 mM Tris-HCl (pH 7.8), 5 mM MgCl, 10 mM 2-mercaptoethanol), 0.5 µl Klenow (1 unit), and 3 µl of [-P]dATP. The probes were incubated for 45 min at 25 °C and purified over a Sephadex G-50 column. Blots were prehybridized for 1 h at 42 °C with hybmix (45% formamide, 0.5% SDS, 10% Denhardt's solution, 10 mM phosphate buffer, 15% dextran sulfate, and 150 µg/ml salmon sperm DNA). Probes (1 10) were denatured by heating at 100 °C for 3 min, added to the hybmix, and incubated at 42 °C for 18 h. The blots were washed and exposed to x-ray film.

Bandshift Analysis

Ribonucleotides were synthesized using the RNA phosphoramidites from Applied Biosystems (Foster City, CA). Oligonucleotides and oligoribonucleotides corresponding to the sequences illustrated were either used as single-stranded DNA or single-stranded RNA or annealed to form a double-stranded DNA product, P-end-labeled with T polynucleotide kinase and purified on Biogel P-60 columns (Bio-Rad). Binding reactions were carried out in 25-µl volumes containing 2 µg of protein from the nuclear extract (unless otherwise stated in the figure legends), 1 µg of poly(dI-dC), varying amounts of unlabeled competitor, and 5 binding buffer (4% glycerol, 1.0 mM MgCl, 50 mM NaCl, 10 mM Tris (pH 7.5), 0.5 mM dithiothreitol). After incubation for 15 min on ice, 4 10 cpm of probe was added and incubated for an additional 30 min. Samples were electrophoresed on a 6 or 8% acrylamide gel (38:1 cross-linking) at 125 V at room temperature with circulating cold water using 0.5 TBE buffer (0.45 mM Tris base, 0.45 mM boric acid, 1 mM EDTA (pH 8.0)). Gels were dried and exposed to x-ray film at -70 °C, except for where the gel was imaged on a PhosphorImager (see Fig. 4 C) (Molecular Dynamics, Sunnyvale, CA).


Figure 4: The mARS is bound by proteins as single-stranded DNA. A, bandshift analysis of the single-stranded, C-rich, sense strand of the mARS. The C-rich strand of the mARS (+861/+880) shown in Fig. 3 was labeled and used as a probe. The molar excess is shown above the competitors. Single-stranded cold competitor is shown where numbers represent starting and ending bp, and Ts are mutant competitors. Addition of 2 µg of GT1-7 protein is shown as a + above the lanenumbers. B, bandshift analysis of the single-stranded, G-rich, antisense strand of the mARS. The G-rich strand of the mARS underlined in Fig. 1 was labeled and used as a probe. The molar excess is shown above the competitors. Single-stranded cold competitor is shown where numbers represent starting and ending bp and As are mutant competitors. Addition of 2 µg of GT1-7 protein is shown as a + above the lanenumbers. C, single-stranded and double-stranded bandshift probes have different mobilities in the absence of protein. The C-rich strand, G-rich strand and the double-stranded mARS were labeled for use as bandshift probes ( lanes1-3, respectively). The probes were separated on a 6% acrylamide gel in the absence of protein. The exact bases used are shown above the lanes. D, bandshift analysis of double-stranded mARS. The double-stranded mARS was labeled, and 5 10 counts were incubated with the amounts of GT1-7 nuclear extracts shown above each lane. The complexes were then separated on a 6% acrylamide gel.



Protein Separation and Southwestern Analysis

Protein was mixed with an equal volume of 2 Laemmli buffer (125 mM Tris-HCl (pH 6.8), 20% glycerol, 4% SDS, 0.005% bromphenol blue, 5% -mercaptoethanol) and incubated for 5 min at 100 °C. Samples were loaded on a 14% Tris-glycine gel (Novex), and run at 150 V for 1.5 h in 1 Tris-glycine SDS running buffer (2.5 mM Tris-Base, 19.2 mM glycine, 0.01% SDS). Protein size was determined using Rainbow high molecular weight range markers (Amersham Corp.). Proteins were allowed to renature by incubation in 50 mM Tris-HCl (pH 7.4) with 20% glycerol for 1 h and transferred to nitrocellulose membrane in 12 M Tris, 96 mM glycine with 20% methanol using 275 mA for 2 h. The membranes were incubated with TBST blocking buffer (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05% Tween 20, 2% skim milk) for 1 h at 25 °C. Single-stranded end-labeled probe (1 10 cpm/ml, prepared as described above) was added and allowed to incubate for 1 h at 25 °C. The blots were washed 2 times with TBST (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05% Tween 20) for 5 min each time and exposed to autoradiography film.

Protein Purification

CNBr-activated Sepharose 4B (Pharmacia Biotech Inc.) was coupled to single-stranded DNA using multimers of the mARS C-rich strand (TCCCCCCACCCACCCCCCCT), G-rich strand (AGGGGGGGTGGGTGGGGGGA), mutated C-rich strand (TCTCCTCACTCACCTCTCCT) or mutated G-rich strand (AGGAGAGGTGAGTGAGGAGA) according to manufacturer's specifications. Protein was mixed with dI-dC in a 4:1 ratio in binding buffer with no dithiothreitol (see ``Bandshift Analysis'') and incubated at 4 °C for 10 min. The protein was applied to either the C-rich or the G-rich column and eluted in 1-ml fractions with increasing amounts of NaCl. Fractions were assayed for DNA binding activity, and active fractions were applied to columns with the mutated DNA to remove nonspecific single-stranded DNA binding proteins. The flow-through was then applied to the C-rich or G-rich column again and eluted with 1-ml fractions using increasing NaCl. The fractions were assayed for DNA binding and protein content. Protein visualization was achieved by silver staining (18) after separation on a Tris-glycine SDS gel as described above.


RESULTS

Mutating the mARS Results in Increased CAT Activity

In order to better define the mARS and demonstrate that the mARS is capable of suppressing a heterologous promoter, a set of constructs was created in which a single copy of the mARS or a portion of the mARS was inserted in the correct orientation in the pBLCAT2 vector. In addition, a set of control constructs was created that contained a mutated mARS or portion of the mARS (Fig. 1 A). The mARS was inserted 3` to the transcription start site of the pBLCAT2 vector, which contains the thymidine kinase promoter, since previous work in this laboratory had shown that the mARS was not functional if placed 5` to the transcription start site (16) . Fig. 1 B shows that the mARS-containing construct +861/+880 was suppressed by 51% as compared with its mutant partner +861/+880T. The two constructs that contained only a portion of the mARS (+872/+880 and +861/+868) had 39 and 11% suppression as compared with their mutants. Previously (16) , a construct that contained the homologous AR promoter had shown a 58% reduction in CAT activity when compared with a mutant construct in which three cytosines had been changed to thymines in the mARS. This indicates that the mARS region from +861 to +880 is capable of suppressing a heterologous promoter in much the same manner as the homologous promoter is suppressed.


Figure 1: The mARS suppresses CAT activity from a heterologous promoter. A, sequences of mARS that were inserted into the pBLCAT2 vector. Construct names ending with a T were mutated. Specific bases that were mutated are shown in boldface. B, the bar graph shows percent activity of each construct. Bars represent the average activity from two separate experiments. The filleddiamonds are the values from experiment one. The opencircles show the values from experiment two. Mutated mARS constructs were assigned a value of 100% as compared with the same unmutated construct. Duplicate plates were used in each experiment. GT1-7 cells were transfected by electroporation and extracts normalized to RSV--GAL.



The mARS Reduces the Amount of AR-initiated RNA

We next investigated the mechanism by which the mARS causes suppression. Because the mARS is located in the 5`-untranslated region of the AR gene, we asked the question, are the mARS effects prior to the level of RNA translation? In order to investigate this, we transiently transfected GT1-7 cells, harvested total RNA, and analyzed the effect of the mARS. Using this method, changes in the amount of CAT RNA should reflect mARS effects. Fig. 2, lane1, shows RNA from GT1-7 cells transfected with vector alone, which does not contain a promoter. The faint band indicates that some nonspecific transcription initiation is occurring or that a small amount of residual plasmid DNA remains. Lane2 shows the amount of total RNA that an mARS-containing construct produced. Lanes3- 5 show RNA produced by constructs from which the mARS has been mutated ( lane3) or deleted ( lanes4 and 5). The increased amounts of transcripts detected in the deletion constructs clearly illustrate that these bands do represent the proper gene products and that the mARS is capable of suppressing the amount of transcripts. As a control, a construct that contained an RSV-driven - gal gene was cotransfected with the CAT vectors, and the blot was reprobed for -Gal RNA. These data illustrate that the mARS is exerting its influence prior to translation.


Figure 2: Northern blot analysis of total RNA harvested from mARS lacking and mARS containing cells. Total RNA was prepared from GT1-7 cells harvested 40 h after they were transiently transfected with mARS-containing constructs ( lane2) and mARS-lacking constructs ( lanes3-5). Constructs transfected are shown above the lanes. Lane1 ( CAT3) RNA from the vector lacking any promoter insert. Lanes2-5 are RNA from pBLCAT3 vector with fragments of mAR promoter inserted into the vector. The exact bases inserted are shown above the lanes where +1 represents the 5`-most site of transcription initiation (13). The -146/+971T ( lane3) contains three point mutations in the mARS that have been previously described (18). Probe was prepared as described using a 300-bp fragment of the pBLCAT3 vector that extended downstream from the multiple cloning region to the EcoRI site. CAT RNA shows amount of RNA produced by constructs. -Gal RNA is from an RSV--Gal vector that was simultaneously transfected and is shown as a control for transfection and loading efficiency.



The mARS Does Not Affect RNA Turnover

In order to further investigate the mechanism of mARS action, we determined the potential effects of the mARS on the half-life of CAT RNA from transiently transfected GT1-7 cells (Fig. 3). A CAT construct that contained the native mARS (-146/+971) or a mutated mARS (-146/+971T) was transfected, and RNA synthesis was halted by the addition of actinomycin D for varying amounts of time (Fig. 3 A). The autoradiogram was analyzed by densitometry and normalized to the 28 S ribosomal band. As had previously been shown, mutation of the mARS caused a marked increase in total RNA. However, analysis of the rate of RNA degradation (Fig. 3 B) indicates that native mARS had a very similar rate of turnover as compared with the mutated mARS. This indicates that the mARS is not functioning by facilitating RNA degradation.


Figure 3: Northern blot analysis of RNA half-life from suppressor containing (-146/+971) and suppressor lacking (-146/+971T) constructs. A, Twenty-four h after transient transfection into GT1-7 cells, actinomycin D was used to block synthesis of new RNA. Time of treatment with actinomycin D ( ActD) is shown above the lanes in hours. The lanes that contain RNA from mARS-containing constructs are designated with a +, and the lanes that contain RNA from mutated mARS constructs are designated with a -. CAT RNA shows amount of construct RNA remaining after varying times of actinomycin D treatment. The 28 S ribosomal band was used for normalization. B, graph of RNA half-life from mARS-containing and mARS-lacking constructs. The graph shows percent of mRNA on a log scale verses time of actinomycin D treatment in hours. Opensquares represent the mARS containing points. Closedsquares represent the mARS-lacking points.



Single-stranded mARS Is Specifically Bound by Proteins

In order to characterize the proteins that had been shown previously (16) to interact with the mARS, we performed bandshift analyses on the mARS DNA. Fig. 4 A shows that when a single-stranded, C-rich, sense strand mARS DNA probe (+861 to +880) is incubated with nuclear extracts from the GT1-7 cells, a specific protein-DNA interaction occurs ( lanes2-5). In addition, an unlabeled, mutated, single-stranded oligonucleotide (shown in Fig. 1) was not able to compete ( lanes6-8), indicating that the protein-DNA interaction is a sequence-specific interaction. Fig. 4B shows that the single-stranded, G-rich, antisense strand is also able to bind specifically to protein ( lanes2-5), although the bound complexes appear to be different from those seen in Fig. 4 A. Lanes6-8 indicate that an unlabeled, mutated, single-stranded oligonucleotide is unable to compete for binding, indicating that this complex is also dependent on specific DNA sequences.

The observation that the mARS was bound by single-stranded DNA-binding proteins led us to question earlier indications that it could be bound as double-stranded DNA (16) . Initial observations indicated that the double-stranded mARS could also bind to protein, but it seemed possible that this was an artifact. Shifts would be created if unannealed single-stranded DNA was present with the double-stranded DNA probes. To confirm that single-stranded DNA was present with the double-stranded DNA probe, we electrophoresed the single-stranded C-rich sense strand, the single-stranded G-rich antisense strand, and the double-stranded mARS on the same gel, in the absence of protein. Fig. 4 C shows that the three different probes have different electrophoretic mobilities and that the double-stranded probe does contain unligated single-stranded DNA. Additional experiments indicated that all double-stranded probes created in this way contained unligated single-stranded DNA (data not shown).

In order to test to see if the mARS could be bound as double-stranded DNA, increasing quantities of nuclear extracts were added to a constant amount of double-stranded mARS. If the mARS is bound as double-stranded DNA, then increasing amounts of shifted probe should be seen. However, the increasing amounts of nuclear extracts did not result in increasing amounts of shifted probe (Fig. 4 D). This indicates that only residual amounts of single-stranded mARS are binding in these experiments. This residual single-stranded sense and single-stranded antisense mARS is again seen below the major band of labeled, double-stranded probe in Fig. 4D, lane1. Based on these data, proteins appear to be able to bind in a sequence-specific manner to either the sense or antisense single-stranded mARS DNA, but the proteins appear to have little or no ability to bind double-stranded mARS DNA.

The mARS Binds Multiple Proteins

In order to investigate the possibility that more than one protein-DNA complex is forming, we performed an additional bandshift experiment with several unlabeled competitors. Fig. 5shows bandshifts with two different labeled probes. In lanes1-3, a single-stranded probe consisting of the C-rich sense strand of the mARS was used. The protein-DNA complex that was formed with this probe was competed away by the C-rich sense strand of the mARS ( lane2). However, the unlabeled single-stranded oligonucleotide, which corresponds to the G-rich antisense strand, was not able to compete for the complex ( lane3). In lanes4-6, a single-stranded probe consisting of the G-rich antisense strand of the mARS was used. However, the protein-DNA complexes formed using this probe were not able to be competed for by the single-stranded C-rich sense strand of the mARS ( lane5). The single-stranded G-rich antisense strand was able to compete for binding with the complexes ( lane6). The difference in the shifted complex seen when lanes4 and 5 of Fig. 5are compared with lane2 of Fig. 4 B appears to be due to the increased amount of protein that was bound and slightly better resolution of the gel in Fig. 4 B. The difference in the mobility of the probe in lane5 of Fig. 5as compared with lanes4 and 6 appears to be due to the single-stranded antisense probe annealing to the unlabeled single-stranded sense competitor. These results confirm that one protein-DNA complex is able to bind to the single-stranded C-rich sense strand of the mARS. A distinctly different protein-DNA complex binds to the single-stranded G-rich antisense strand of the mARS.


Figure 5: Bandshift analysis showing multiple proteins bind to the mARS. Bandshifts were performed using 0.5 µg of GT1-7 nuclear extract for lanes1-3 and 2.0 µg of GT1-7 nuclear extract for lanes4-6. Extracts were incubated with 5 10 counts of the mARS oligonucleotides and separated on a 6% acrylamide gel. Two different probes were radioactively labeled and are shown above the arrows. Single-stranded sense mARS ( mARS (C)) was used for lanes1-3, and single-stranded antisense ( mARS (G)) was used for lanes4-6. Cold competitors were used in 50-fold molar excess.



The mARS Binds Proteins from Multiple Tissues

In order to further characterize the suppressor proteins, we performed bandshifts using nuclear extracts from multiple tissues. Fig. 6A shows that the single-stranded C-rich sense strand of the mARS interacts with proteins from all tissues examined. The mARS proteins from most tissues formed similar complexes. However, the complex formed with the nuclear extract from the submandibular gland was smaller. The single-stranded G-rich antisense strand of the mARS also interacted with proteins from all tissues examined (Fig. 6 B) but with different complexes from those seen with the single-stranded C-rich sense strand of the mARS. These data demonstrate that the mARS proteins are found in many different tissues and suggest that the two strands of DNA may interact with different proteins.


Figure 6: Proteins from multiple tissues bind to the mARS. A, bandshifts of the single-stranded, C-rich, sense strand of the mARS bound by proteins from multiple tissues. Bandshift analysis was performed as described above using the C-rich sense strand of the mARS as the labeled probe. Specific tissues that the nuclear extracts were made from are shown above the gel. B, bandshifts of the single-stranded, G-rich, antisense strand of the mARS. Bandshift analysis was performed as described above except that the labeled probe was produced using only the G-rich antisense strand of the mARS. Specific tissues that the nuclear extracts were made from are shown above the gel.



Determination of the Size of the Proteins That the mARS Binds to

To determine the size of the different mARS proteins, Southwestern analysis and protein purification were performed. Southwestern analysis allows one to analyze proteins that have been size-fractionated on an SDS-polyacrylamide gel for their ability to bind to a radioactive DNA probe. The proteins that bind to the single-stranded C-rich sense strand of the mARS and the proteins that bind to the single-stranded G-rich antisense strand of the mARS were examined, and the results are shown in Fig. 7. Southwestern analysis of unpurified protein from the liver demonstrated that the sense strand binds two different sized proteins ( lane1). The major protein bound is 52 kDa, and the minor protein bound is 90 kDa. A purified fraction from the liver was obtained using a DNA affinity column specific for the sense strand of the mARS. On a silver-stained gel, the purified fraction exhibited a single protein band of approximately 52 kDa ( lane2). This purified fraction retained the ability to form a complex with the single-stranded C-rich sense strand of the mARS as shown by bandshift analysis ( lane3). A Southwestern blot of unpurified protein from the spleen using the antisense mARS as a probe is shown in lane4. Two bands were observed at approximately 35 and 28 kDa. These proteins were then purified using a DNA affinity column specific for the antisense strand of the mARS. Silver staining revealed a doublet of 33 and 35 kDa ( lane5). The purified proteins retained the ability to bind to the antisense DNA as shown by a bandshift assay ( lane6). These results indicate that at least one protein of approximately 52 kDa is capable of binding to the sense strand of the mARS and that a doublet of 33 and 35 kDa is capable of binding to the antisense strand of the mARS.


Figure 7: Protein purification and size determination of the mARS proteins. Southwestern analysis ( lanes1 and 4) was performed using 2 µg of total nuclear extract from liver ( 1) or spleen ( 4). Single-stranded probes corresponding to the sense ( 1) and antisense ( 4) mARS were labeled and used as probes. Purified protein is shown as silverstained fractions from DNA affinity columns ( lanes2 and 5), which were specific for the single-stranded sense ( 2) and single-stranded antisense ( 5) mARS. The fractions were generated by using increasing amounts of NaCl to elute bound protein. Nonspecific DNA binding proteins were removed by applying the extracts to DNA affinity columns that contained single-stranded mARS with point mutations inserted. Bandshifts ( lanes3 and 6) were performed as described previously using the same fractions as shown in lanes2 and 5.



RNA Corresponding to the mARS Can Be Bound by Protein

Because the mARS is found in the 5`-untranslated region of the AR gene, we asked if RNA from the mARS could be bound by protein. Fig. 8shows a bandshift experiment in which a radioactively labeled RNA probe that corresponds to the mARS sense strand from +861 to +880 is bound by protein(s) from the GT1-7 cells ( lane2). One-hundred-fold cold +861/+880 RNA competitor was able to compete for RNA binding ( lane3), but the mutated +861/+880 RNAU was not able to compete for binding ( lane4), indicating that the protein-RNA interaction is a sequence-specific interaction. In order to determine if the same proteins that bound the mARS RNA could bind to the mARS DNA, we used the single-stranded, C-rich, sense strand of DNA and the single-stranded, G-rich, antisense strand of DNA as cold competitors for the RNA binding. The data indicate that the C-rich, sense strand of DNA is able to compete for binding ( lane5) but that the G-rich, antisense strand of DNA is not able to compete for binding ( lane6). This experiment indicates that the mARS RNA can be bound by protein and that the protein may be identical to that bound to the C-rich DNA strand of the mARS.


Figure 8: Bandshift analysis of proteins binding to the mARS RNA Bandshifts were performed using 0.4 µg of GT1-7 nuclear extract. Extracts were incubated with 5 10 counts of the labeled mARS oligoribonucleotide and separated on a 8% acrylamide gel. The sequence of the unmutated mARS RNA ( +861/+880 RNA) was UCCCCCCACCCACCCCCCCU and the mutated mARS RNA ( +861/+880 RNA U) was UCUCCUCACCCACUCUCUCU. The sequences of the cold competitor DNA is shown in Fig. 3. Specific cold competitors used are shown above each lane.




DISCUSSION

The 5`-flanking region of the mouse androgen receptor has been shown to contain a region that is capable of suppressing activity from both the AR gene promoter and the heterologous thymidine kinase promoter. The region appears to function by suppression of AR transcription, since it is not dependent on translational effects and does not facilitate RNA degradation. We have termed this region the mARS.

Characterization of the mARS has shown that it is bound by multiple, sequence-specific, proteins. Some of these proteins are capable of binding the single-stranded C-rich sense strand of the mARS DNA or RNA, and a different protein(s) binds to the single-stranded G-rich antisense mARS DNA. Proteins capable of specifically binding to the mARS are found in all tissues tested to date. However, these tissues differ in quantity of binding and appear to contain different proteins or protein complexes capable of binding to the mARS. Thus, it is possible that the mARS may have tissue-specific functions. Southwestern analysis and protein purification have revealed a 52-kDa band that binds to the C-rich sense strand of the mARS and a doublet of 33 and 35 kDa that binds to the G-rich antisense strand. The complexes are identical in size to those seen using unpurified nuclear extracts. Interestingly, increasing amounts of purified liver extract caused the lower band of the complex formed with the C-rich sense strand (Fig. 7, lane3) to disappear, suggesting that the two complexes may represent monomer and dimer complexes (data not shown).

Information about the specific proteins that bind to the mARS is of great interest. It is exciting to note that a second suppressor with some sequence similarity has been shown to exist upstream of the promoter-1 in the AR gene (19) . Additional experiments may determine the interactions of these two suppressors. Reports exist in the literature of other single-stranded DNA binding proteins. A very large family of evolutionarily conserved Y-box proteins have been well characterized (20) . Analysis of the literature has revealed three potential proteins that bind to sequences similar to the mARS. The SW2 protein is a negative regulator of catalase gene expression (21) , and the heterogeneous ribonucleoprotein particle proteins K and A1 serve as positive transcriptional elements for the c- myc gene (22) . All of these proteins function by influencing transcription from a location that is 5` to transcription initiation. Protein sequence or antibodies should help determine whether any of these proteins are involved in the AR gene suppression.

The mechanism by which these proteins regulate AR transcription is open to speculation. Two potential models of transcriptional suppression are possible, based on the current data. One model involves inhibition of transcription initiation by binding of the protein(s) to the mARS. The DNA must then bend back so that the mARS protein(s) could interact with other members of the transcription complex to prevent transcription from initiating. Another model (Fig. 9) involves blockage of transcription elongation by the protein(s) binding to both the C-rich strand of the mARS and the G-rich strand of the mARS. This model is favored by us for two reasons. First, it accounts for the fact that in previous experiments (16) the mARS is capable of suppressing transcription only if it is located 3` to transcription initiation. Second, it explains the need for two distinct sets of single-stranded DNA binding proteins. It is possible that as the DNA is transformed from a double-stranded state to a single-stranded state during the process of transcription, the mARS binding proteins may be able to interact with the DNA and form a roadblock to transcription elongation.


Figure 9: Potential model of mARS action. Stylized model of transcription attenuation by the mARS. The mARS bpC (mouse androgen receptor suppressor binding protein C) represents protein(s) capable of binding to the C-rich sense strand of the mARS DNA ( double- strandeddarkgrayribbon) or the mARS RNA ( single- strandedlightgrayribbon). The mARS bpG (mouse androgen receptor suppressor binding protein G) represents protein(s) capable of binding to the G-rich antisense strand of the mARS. Dashed, two- headedarrows indicate the potential for mARSbpG protein sequestration by mARS RNA and subsequent transcription elongation. Single- headedsolidarrow with X represents elongation blockage. Single- headedsolidarrow without X represents transcription elongation. The largeoval represents the general transcription apparatus.



Additional information about the potential mechanism of mARS suppression is gained by the knowledge that both the C-rich sense strand of the mARS DNA and the mARS RNA can compete for the same protein(s). In this way, the formation of the roadblock may be directly affected by the amount of AR RNA present (Fig. 9). Other laboratories have shown that some single-stranded DNA binding proteins are capable of binding to RNA and sequestering proteins that negatively regulate transcription (23) . This could be a method for ensuring that transcription is maintained in a situation where the protein is a transcriptional suppressor. Further study of the mARS proteins should yield interesting details about this newly emerging area of transcriptional regulation.

In conclusion, these studies further support the novel observation that the mouse androgen receptor has a suppressor region located in the 5`-untranslated region of the gene. We have termed this region the mARS and have shown that the mechanism of suppression is transcriptional. The proteins involved in the suppression exhibit several interesting properties. Further study of these proteins should yield additional information about regulation of the AR specifically and about transcription in general.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants DK47592, HD09140, and CA58225. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be sent addressed: Depts. of Urology, Biochemistry, and Molecular Biology, Mayo Clinic, Guggenheim 17, Rochester, Minnesota 55905. Tel.: 507-284-8553; Fax: 507-284-2384.

The abbreviations used are: AR, androgen receptor; bp, base pair(s); CAT, chloramphenicol acetyltransferase; mARS, mouse AR suppressor; MOPS, 4-morpholinepropanesulfonic acid; RSV, Rous sarcoma virus.


ACKNOWLEDGEMENTS

We thank Marceil Blexrud for excellent technical assistance in conducting the experiments for this manuscript. We also thank Dr. Leen Blok for discussions about and critical review of this manuscript.


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