A Consensus DNA-Binding Site for the Androgen Receptor

Peter

J. Roche*,

Sue A. Hoare,

and Malcolm

G. Parker

Molecular Endocrinology Laboratory Imperial Cancer Research Fund London, United Kingdom WC2A 3PX

We have used a DNA-binding site selection assay to determine a consensus binding sequence for the androgen receptor (AR). A purified fusion protein containing the AR DNA-binding domain was incubated with a pool of random sequence oligonucleotides, and complexes were isolated by gel mobility shift assays. Individually selected sites were characterised by nucleotide sequencing and compiled to give a consensus AR-binding element. This sequence is comprised of two 6-basepair (bp) asymmetrical elements separated by a 3-bp spacer, 5’GGA/TACANNNTGTTCT-3’, similar to that described for the glucocorticoid response element. Inspection of the consensus revealed a slight preference for G or A nucleotides at the +l position in the spacer and for A and T nucleotides in the 3’flanking region. Therefore, a series of oligonucleotides was designed in which the spacer and flanking nucleotides were changed to the least preferred sequence. Competition experiments with these oligonuclueotides and the AR fusion protein indicated that an oligonucleotide with both the spacer and flanking sequences changed had greater than 3-fold less affinity than the consensus sequence. The functional activity of these oligonucleotides was also assessed by placing them up-stream of a reporter gene in a transient transfection assay and correlated with the affinity with which the AR fusion protein bound to DNA. Therefore, sequences surrounding the two 6-bp half-sites influence both the binding affinity for the receptor and the functional activity of the response element. (Molecular Endocrinology 6: 2229-2235,1992)

ment is largely determined by the N-terminal zinc finger, although the C-terminal finger is also necessary for DNA binding (4-7). Two classes of steroid receptors have been described which differ in their DNA-binding specificity. The glucocorticoid receptor is a member of the first class, which recognizes a 15-basepair (bp) consensus sequence, while the second class, which includes the estrogen receptor, recognizes a similar, yet distinct, 13-bp response element (8-10). Although a clear consensus sequence has not been defined for the androgen receptor, a glucocorticoid response element (GRE) can mediate induction by androgens (11-13). A small number of cellular genes have been characterized that are regulated by androgens. Some of the best studied include the C3 (14-18) and prostate-specific antigen genes (19, 20). Two response elements from these genes, AGTACGtgaTGTTCT and AGAACAgcaAGTGCT, respectively, which confer androgen regulation to a reporter gene, are similar to the GRE consensus. At this stage, however, it is uncertain whether there is an AR recognition sequence that constitutes a specific androgen response element (ARE) distinct from the GRE. In an effort to understand the exact sequence requirements of an ARE, we have examined the nucleotide sequences that bind the AR in a DNA-binding site selection assay (21-23). In this assay we have used a purified AR fusion protein that was expressed in E. co/i. This protein interacts specifically with a putative ARE in the C3 gene (24). The source of potential binding sites was a pool of random sequence oligonucleotides, and complexes were purified by gel mobility shift assays. Individually selected sites were characterized by nucleotide sequencing and binding analysis, which allowed us to determine a consensus binding sequence for the AR.

INTRODUCTION The androgen receptor (AR) is a member of the steroid hormone receptor family that can regulate gene transcription by interacting with specific DNA elements (l3). These receptors share a highly conserved DNAbinding domain, which forms two zinc fingers. The specificity of a receptor for its hormone respone ele088e-8809/92/2229-2235$03.00/0 Molecular Endocrmolqy Copyright 0 1992 by The Endcmne

RESULTS Selection

of AR-Binding

Sites

A fusion protein containing the DNA-binding domain of the AR and a random oligonucleotide pool have been used in a binding site selection assay. The recovery of AR-binding sites using the assay is shown in Fig. 1. As the binding site selection is repeated, the probe oligo-

Scxxty

2229

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Vol6 No. 12

MOL ENDO. 1992 2230

Protein

AR fusion Cycle

II 0

1

2

3

4

0

1

2

A 3

4

Fig. 1. Gel Shift Analysis of Selected AR-Binding Sites DNA selected at each round was used as probes in a gel mobility shift assay with the indicated proteins. The number of selection cycles is shown above each lane (0 denotes Bind 76 oligonuclectide). In this assay, binding reactions contained either 50 ng AR fusion protein or protein-A with 15 ng oligonucleotide probe. Exposure was for 1.5 h with an intensifying screen.

nucleotide pools become enriched in sequences that form complexes with the AR fusion protein. Enrichment occurs in this assay because there is competition between binding site oligonucleotides for a limiting concentration of protein. There was no binding of selected sequences with protein-A that was purified from an E. co/i culture harboring the parental pRIT2T vector. This indicates that the binding activity of the probes was specific for the DNA-binding domain of the AR. Sequence

and Binding

Analysis

of Selected

Sites

The DNA recovered after four rounds of selection (Fig. 1) was subcloned into pBLCAT2, and 24 individual clones were subjected to nucleotide sequence analysis (Table 1 A). Initially, their sequences were compared in both orientations, and a consensus sequence Y-G/ AGA/TACANNNTGTT/ACT/C-3’ was derived from the compilation data (Table 1 B). When each sequence was orientated to maximize the match of the 3’-half-site with the sequence TGTTCT, a second compilation table was obtained to derive consensus sequence comprised of two 6-bp asymmetrical elements separated by a 3bp spacer, 5’-GGA/TACANNNTGTTCT-3’ (Table 1 C). Surprisingly, the two 6-bp elements do not have complete dyad symmetry, as might be expected if a homodimer was binding to the two half-sites in an equivalent manner. Inspection of this consensus reveals a preference for G or A nucleotides at the +l position in the 3bp spacer and a slight A/T preference flanking the 3’half-site.

We also examined the sequences of sites recovered from complexes formed by DNA selected after two rounds (Fig. 1, cycle 2). Of a total of 26 clones analyzed, 10 had homology to the consensus sequence, 15 contained a 6-bp half-site sequence, and one clone had no similarity to the consensus. The consensus sequence derived from the 10 clones that contained two half-sites is 5’-GGA/TACANNNTGTTCT-3’ (Table 1D). This sequence does not show any preference for G or A nucleotides at the 1 position or for A/T nucleotides in the 3’-flanking region, unlike that derived from round 4. A number of individual binding sites were tested for complex formation with the AR fusion protein. Four binding sites were examined in detail, three from round 4 and one from round 2. The results indicate that there was a significant range of binding affinities for some of the sites that were isolated (Fig. 2). Also, competition with a GRE oligonucleotide showed that binding of the AR fusion protein to the individual sites is specific to the GRE-like consensus sequence. Therefore, the binding site sequences from round 4 that were used to construct the consensus exhibited a range of affinities and were not solely of high affinity. In addition, we examined the DNA-binding activity of the 15 sites that contained only a 6-bp half-site sequence and found that they all exhibited very weak binding activity (data not shown). Nucleotides Flanking the Half-Sites Influence Binding Affinity and Functional Activity An examination of the consensus sequence for the ARbinding site (Table 1, round 4) revealed a slight preference for G nucleotides in the 3-bp spacer region, particularly at the 1 position and A/T nucleotides in the 3’position (positions 8-l 1). Therefore, a series of oligonucleotides was constructed in which the central and flanking nucleotides were altered, and their DNA-binding activities were compared with the results of competition experiments (Fig. 3). An oligonucleotide (ARE2) in which the spacer nucleotides were changed to the least prefered sequence competed with almost the same affinity as the consensus ARE oligonucleotide. However, an oligonucleotide (ARE-3) with the flanking nucleotides changed to the least prefered sequence had approximately 3-fold less affinity than the consensus oligonucleotide. Likewise, an oligonucleotide (ARE-4) with both the spacer and flanking sequences changed had greater than 3-fold less affinity than the consensus ARE. These results indicate that sequences flanking the two 6-bp half-sites influence the binding affinity for the receptor. We next assessed the functional activity of the four binding sites up-stream of the thymidine kinase promoter in transiently transfected ZR-75.1 cells, which contain endogenous ARs. These results are shown in Fig. 4. Promoter activity was stimulated up to 4-fold by ARE-I, the consensus binding site gtctGGTACAgggTGTTCTtttt. Substitution of the spacer nucleotides in ARE-2 or flanking sequences in ARE-3 resulted in tran-

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CTCC GCGT TGTC GTCG TGTC TGTC GACT TTAT TCTT GATA AGGG CATA CGAG CGCA GTCA AACT GGGC TGCA GTAA GTTG GTCA GTGT CGGA TAAC

15 12 9 12

13 10 16 7

8 13 16 11

aligned

10 3 6 5

10 2 7 5

2

9 5 8 2

11 2 9 2

5 4 7 8

6 4 7 7

-8

-8

4 8 6 6

5 7 8 4

G

19 3 1 1

G

16 4 3 1

G

23 0 1 0

-6

G

23 0 1 0

-6

G

45 0 3 0

-6

A/T

40 8 8 4

TA

40 6 12 2

A/T

6 19 16 7

A

18 4 2

-4

19 4 1

-4

A

0 41 5 2

-4

A

13 30 4 1

-2 11 4 15 18

0 13 11 11 13

A

C

8 3 7 6

9 3 5 7

0

10 7 4 3

0

11 6 5 2

12 8 1 3

11 10 1 2

18 15 4 11

T

0 2 17 5

2

T

0 2 16 6

shown

T

0 1 23 0

4

T

1 1 22 0

4

T

2 5 41 0

4

(A),

7 16 19 6

C

T

1 2 17 4

T

1 0 16 7

T/C

2 3 20 23

2 7 9 6

8

4 5 10 5

8

8 13 17 10

8

3 9 9 3

4 9 9 2

11 16 13 8

5 8 7 4

10

5 7 10 2

10

7 16 12 13

10

7 2 10 5

7 3 9 5

12 9 12 15

alignedaroundthetwo 6-bp elements, whichare boxed. (B). The sequenceswerethenorientatedtomaximizethematchofthe3’-halffrom10 sites isolated fromround 2is also shown(D).

T

0 2 0 22

6

C

T

3 8 11 2

0 2 0 22

6

C

0 3 0 45

6

5 4 13 2

T/A

compilation symmetry.

G

24 0 0 0

G

24 0 0 0

G

T

2

48 0 0 0

1 4 30 13

2

rounds of selectionare sequencebasedon48sequences sequence (C). Asimilar relativetothecenterofdyad

four

8 13 2 1

-3

A

7 14 2 1

-3

0 0 0 24

C

0 0 0 24

3’-half-SitewiththesequenceTGTTCT)

C

0 0 0 48

The sequences of 24 clonedAR-binding sitesderivedby compiled initially inbothorientationstoderiveaconsensus SitewiththesequenceTGTTCTtoderivea secondconsensus compilationtablerefertothepositions ofnucleotides

CO”Se”S”S

G A T C

CO”Se”S”S D Round 10

G A T c

10

G/A

23 20 3 2

orientations)

AACT CTCC TTTG TAAT TACT ACTT TAGA TTTT AGTC ATCG TTAG CTTG TTTT GTAT GAGG ACAT AGGG CCGT CAAC CATC TTGA TAAG AAAT CGAC

for the AR

CGTACT TGTTCT TGTACT CGTACG TGTACT TGTTCT TGTGCT TGTTCC AGTCCT TGTACA TGTTCC TGTTCT TGTAAT CGTTAT TGTGCT TGTACT CGTTCT TGTGCT CGTTCT AGTTCT TGATCC TGTTCT TGTCCC TGTACA

Sites

inboth

10 17 13 8

-8

C Round 4 (sequencesalignedtomaximizethematchof

CO”Sl?“S”S

G A T C

10

CCG AAA GGG TAT GAG TTA CGG TGA ATG TGG GGG TGA CGA GGA GAC CAG GGA GGC ACG CTA TAG GAG CTG TCC

Binding

GGAACG AGAACG GGTACA GGTACG GGAACA GGAACA GGGACA TTCACA GGACCG GGCACA GGGTCA GGAACG GGTTCA GGGACT GCTACG AGCACA GGATCA GGGACA AGCACG GGTACT GGTTCA CGTACA GGTACC GCACCG

1. Selected

B Round 4 (sequences

A 4.1 4.2 4.3 4.4a 4.4b 4.6 4.7 4.8 4.9 4.10 4.12 4.13a 4.13b 4.14 4.17 4.20 4.21 4.29 4.30 4.36 4.37 4.41 4.46 4.22

Table

Thenumbers

These

above

sequences

each

were

MOL ENDO. 1992

Vol6 No. 12

2232

BC\IDING

416

4.17

2.43

4.29

v

* 4.6

GGAACA;TTA;TGTTCT

4.17

GGTACGlGAClTGTGCT GGGACA:GGC;TGTGCT GGAACAiAAAlTGTCCC

4.29 2.43

GRE

AGAACA

CAG

TGTTCT

2. Characterization of Individual Binding Sites Binding reactions contained 15 ng AR fusion protein and 2 ng of the indicated oligonucleotide probe. The competition oligonucleotide was a consensus GRE, which was used at 40and 400-fold molar excesses. The sequences of the individual sites and that of the the GRE are shown beneath the gel mobility shift assay. Fig.

scriptional activation of 2- to 3-fold, while substitution of both sequences in ARE-4 resulted in transcriptional activation of less than 2-fold. These results approximately parallel the activity with which ARs bind to the four oligonucleotides ARE-l to ARE-4.

ARE-1

GTCT

GGTACA

GGG

TGTTC;

TTTT

ARE-2

GTCT

GGTACA

ACT

TGTTCT

TTTT

ARE-3

TCACGGTACAGGGTGTTCTGCCA

ARE-4

TCACGGTACA

Fig. 3. Competition Experiment with Four Different ARE Oligonucleotides In this gel mobility shift assay, all binding reactions contained 15 ng AR fusion protein and 1 ng labeled ARE-l oligonucleotide probe. Reactions contained either no competitor (indicated by 0) or increasing amounts of the competitor oligonucleotides ARE 1, ARE 2, ARE 3, or ARE 4, as indicated. Competitor DNA was at 40-, 120-, and 400-fold molar excesses. The sequence of the ARE oligonucleotides is shown beneath the gel mobility shift assay.

CONSTRUCT

CATlLUC 0

DISCUSSION

We have used a DNA-binding site selection assay to determine a consensus binding sequence for the AR. The consensus sequence is a 15-bp element comprised of two imperfect 6-bp indirect repeats 5’-GGA/TACANNNTGTTCT-3’. This sequence is almost identical to the consensus GRE, 5’-GGTACANNNTGTTCT-3’, which has been derived from numerous glucocorticoidresponsive genes (2). The recognition of a GRE-like sequence by the AR probably reflects the highly conserved P and D boxes in the DNA-binding domains of the ARs and glucocorticoid receptors (7). The P box recognizes a specific half-site DNA sequence, while the D box recognizes the spacing between half-sites. The P box is identical in the two receptors, whereas there is one amino acid difference in the D box. The ARE consensus sequence does not have complete dyad symmetry with respect to the two 6-bp elements, and there is a preference for G or A nucleotides at the +l position in the 3-bp spacer. This asymmetry suggests that a homodimer of the receptor is not

ACTTGTTCTGCCA

100

200

300

400

500

INDUCTON FACTOR

ARE-l

;

3.7

ARE-2

;

2.8

ARE-3

;

2.3

ARE-4

;

1.5

Effect of Surrounding Sequences on ARE Activity The four oligonucleotides (ARE 1-4) used in competition experiments (see Fig. 3) were subcloned into the Xbal site of the tyrosine kinase promoter CAT reporter gene (pBLCAT2). After transfection into ZR75-1 cells, they were maintained in the presence (+) or absence (-) of 2.5 x lo-* M dihydrotestosterone. Cell-free extracts were assayed for CAT activity and luciferase (LUC) to correct for differences in transfection efficiency. Fig. 4.

binding to the two half-sites in an equivalent manner. These data are consistent with studies which indicate that steroid receptors bind to DNA as dimers in a cooperative manner (25-28). Thus, if cooperative binding occurs, it is intrinsic to the homodimer structure that recognizes the DNA sequence. This is because the

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AR-Binding Sequence

in vitro selection assay was performed with purified fusion protein; thus, there is no influence from other cellular proteins that could alter the specificity of the receptor for its binding site. The optimal asymmetric binding site derived from this in vitro study correlates extremely well with functional data from the mouse mammary tumor virus response elements (13, 29), which indicates that a perfect inverted repeat of the half-site sequence TGTTCT does not give maximum activity as a steroid response element. An examination of the frequency of specific nucleotides in the consensus sequence reveals that certain positions are tightly constrained, whereas others allow more flexibility. The positions 3,-3; 4,-4; and 6,-6 are highly constrained, whereas 2,-2 and 7,-7 are moderately flexible, and 5-5 shows the greatest flexibility. These observations are consistent with data from the report of Nordeen et al. (29), who examined the functional significance of individual substitutions within the GRE. Thus, the importance of certain nucleotides in the ARE, as determined by these in vitro binding studies, correlates well with functionally important nucleotides in the GRE. Two response elements have been characterized from the androgen-responsive genes C3 and prostatespecific antigen, which confer androgen regulation to a reporter gene (14, 20). These elements, AGTACGtgaTGTTCT and AGAACAgcaAGTGCT, are similar to the consensus AR-binding sequence GGA/TACANNNTGTTCT, which was determined in this study. The present work suggests that novel binding sites specific for ARs probably do not exist. One mechanism by which specific hormone responses could be mediated in cells containing more than one type of receptor is suggested by the observation that the activity of steroid receptors can be regulated by the c-jun/c-fos family of proteins (30-33). For example, c-jun has been reported to stimulate transcription from the mouse mammary tumor virus promoter in the presence of the AR, but not in the presence of progesterone or glucocorticoid receptors (34). An analysis of ARES in which the nucleotides surrounding the two half-sites were altered reveal that these sequences influence the binding affinity for the receptor. Also, when these altered elements were assessed for functional activity in ZR75-1 breast cancer cells, they showed a corresponding decrease in testosterone induction. While nucleotides flanking the halfsites do not influence the specificity of the receptorDNA interaction, previous work has shown that flanking sequences are, in fact, necessary for efficient binding of the glucocot-ticoid receptor to its response element (35). It was found that short oligonucleotides containing the 15-bp GRE with l-3 flanking bp on each side were bound with very low affinity. The same GREs, when positioned in the center of a larger DNA fragment (4050 bp), showed high affinity for the receptor. Thus, it was concluded that the nonconserved DNA sequences flanking the GRE contribute significantly to the free energy of receptor binding to DNA. It appears that the

2233

overall nucleotide sequence surrounding the half-sites is important, because individual specific nucleotides can be mutated with negligible effects on the functional activity of the response element (29). In our study it was found that changing the sequence surrounding the half-sites to the least preferred nucleotides resulted in 3-fold lower affinity compared to that of the consensus binding sequence. It is quite possible that the sequences surrounding the two half-sites could influence binding affinity by confering a specific DNA conformation on the response element which favors stability of the receptor-DNA interaction. This DNA conformation could relate to the bending of the DNA helix or the width of the minor groove. Another explanation might be that A-T forms a less rigid duplex than G-C in the flanking region of the binding site and, consequently, may allow better binding to the consensus sequence itself. When a number of ARES have been identified in natural promoters, it will be interesting to see if there is a preference for spacer or flanking nucleotides. Inspection of the ARES in the genes for C3 (1) and prostatespecific antigen shows that they do not conform to the preferred selected binding site, but obviously more ARES will have to be identified and characterized before firm conclusions are drawn.

MATERIALS Expression

AND METHODS and Purification

of the AR Fusion

Protein

An expression vector containing a fragment of the rat AR fused to protein-A was obtained from G. Verhoeven’s laboratory. This construct has the DNA-binding domain (amino acids 533-637) of the rat AR inserted into the polylinker site of the prokaryotic expression vector pRIT2T. The resulting fusion protein is synthesized under control of the phage PR promoter, which is temperature inducible in E. co/i cl857 strains. Expression and purification of the fusion protein were performed as described by De Vos and co-workers (24). The purified protein was greater than 90% pure, as judged by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and silver staining, and was stored frozen in 25 mM HEPES-KOH (pH 7.6), 40 mM KCI, 0.1 mM EDTA, 10% glycerol, and 1 mM dithiothreitol. Binding

Site Selection

Assay

The binding site selection assay used in this work is based on the method of Pollock and Treisman (21). An oligonucleotide pool was used which comprised a random 26-bp sequence flanked by two 25nucleotide polymerase chain reaction (PCR) primer sites (Bind 76). The binding conditions were similar to those used for gel mobility shift assays (35). The receptorDNA complex was separated from unbound DNA on a nondenaturing 6% polyacrylamide gel and excised, and the DNA was eluted. The eluted DNA was then used as a template for PCR, and the amplified DNA was subsequently used for another binding reaction. This procedure was repeated until sufficient enrichment of specific binding sites was attained. Thus, the assay for DNA enrichment and the purification step were performed in a single gel mobility shift assay. In the first round of binding site selection, it was difficult to detect receptor-DNA complexes due to their very low abundance. Therefore, the mobility of the receptor-DNA complex

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MOL 2234

END0.1992

Vol6No.12

on a polyacrylamide gel was estimated by using a GRE 76’mer oligonucleotide, with the AR as a marker. It was known that the AR fusion protein could bind to a GRE sequence (21). After the first round of selection, receptor-DNA complexes could be readily detected on a gel mobility shift assay. Oligonucleotides

Reactions

Binding reactions (30 ~1) contained 20 mM HEPES (pH 7.9) 100 mM KCI, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.1% Nonidet P-40, 15% glycerol, 10 pg acetylated BSA, 1 rg poly(dldC)-poly(dldC), 100 ng AR fusion protein, and 20 ng random oligonucleotide. Complexes were allowed to form at 20 C for 30 min. Protein-DNA complexes were separated from unbound oligonucleotide probe by a gel mobility shift assay. Gel Mobility

C, 1 min; 62 C, 1 min; and (Techne Programmable Dri76’-mer PCR product was polyacrylamide gel; eluted mM EDTA, and 0.1% SDS;

Sequence

Sites

and Probes

Random sequence oligonucleotide Bind 76, 5’-CACGTGAGTTCAGCGGATCCTGTCG(N)26GAGGCGAATTCAGTGCAACTGCAGC3’(300ng), was rendered double stranded using 200 ng F25 (5’-GCTGCAGTTGCACTGAATTCGCCTC3’) in a 20-~1 reaction containing 10 mM Tris-Cl (pH 7.8); 50 mM NaCI; 10 mM MgCIP; 1 mM dithiothreitol; 20 &i [&‘P] deoxy-CTP (3000 Ci/mmol); 50 PM each of deoxy-ATP, deoxyGTP, and deoxy-TTP; 20 PM deoxy-CTP; and 1.0 U Klenow fragment. For PCR reactions, the F25 primer was used in conjunction with R25,5’-CACGTGAGTTCAGCGGATCCTGTCG-3’. A 76’-mer double stranded probe that contained a GRE-bindina site was produced bv annealing the olioonucleotide 5’-CG?AGTACGACTCACTCTAGAGTCCTGATCAGAACACTGTGTTCTGACTTTCCTGGTCGACTTCTATAGTGTCACC3’ with an oligonucleotide of reversed and complementary sequence, which was then labeled with [u-32P]ATP and T4 polynucleotide kinase. A series of double stranded ARE oligonucleotides was used as competitors in gel mobility shift assays and pBLCAT2 reporter plasmids. The sequences of these oligonucleotides were: ARE 1, 5’-CTAGAAGTCTGGTACAGGGTGTTCTTTTTGCA-3’; ARE 2, 5’-CTAGAAGTCTGGTACAACTTGTTCTTTTTGCA-3’; ARE 3, 5’-CTAGAATCACGGTACAGGGTGTTCTGCCAGCA-3’; and ARE 4, 5’-CTAGAATCACGGTACAACTTGTTCTGCCAGCA-3’. Complimentary oligonucleotides were made for each of the above sequences, so that when they were annealed, they had Xbal overhanging ends. For reporter plasmids in ZR75-1 cells, the double stranded oligonucleotides were subcloned into the Xbal site of pBLCAT2. The resulting recombinants were sequenced by the chain termination method. Binding

was carried out for 15 cycles (94 72 C, 1 min) in a thermal cycler Block PHC-1, Princeton, NJ). The purified on an 8% nondenaturing into 0.5 M ammonium acetate, 1 and ethanol precipated.

Shift Assays

Receptor-DNA complexes were resolved on 6% polvacrvlamide (38:l cross-linked) gels in 0.5 x TBE buffer (89 mu Tris-Cl. 89 mM boric acid. 4 mM EDTA DH 8.3). run at 10 V/ cm. Analytical gels were dried, unfixed, on 3 MM paper (Whatman, Clifton, NJ) and autoradiographed. Preparative gels, which were not dried, were subjected to autoradiography for several hours. Oliqonucleotides in complexes were recovered from these gels by excising the band and eluting the DNA into 0.5 M ammonium acetate, 1 mM EDTA, and 0.1% SDS for 16 h at 37 C. Eluted DNA was ethanol precipitated and resuspended in 20 ~1 10 mM Tris-Cl (pH 7.6) and 0.1 mM EDTA. PCR Amplification The recovered DNA (l-4 ~1) from the gel mobility shift assay was amblified in a 20~1 reaction containinq 10 mM Tris-Cl (pH 8.5); 50’mM KCI; 1.5 mM MgCI,; 0.1 mg/ml BSA; 50 FM each of deoxy-ATP, dexoy-GTP, and deoxy-TTP; 20 /IM deoxyCTP; 10 &i [&‘P]deoxy-CTP (3000 Ci/mmol); 100 ng each of F25 and R25 primers; and 2 U Taq polymerase. Amplification

Analysis

of Selected

Selected DNA was PCR amplified and purified as previously described. This isolated DNA was end-repaired with Klenow fragment, phosphorylated with T4 polynucleotide kinase, and ligated into the Xbal site of pBLCAT2, which had been rendered blunt ended with Klenow fragment. Ligated DNA was then transformed into E. co/i DH5 cells. Miniprep DNA was prepared and sequenced by the chain termination method, using Sequenase (U.S. Biochemical Corp., Cleveland, OH). Cell Culture

and Transient

Transfection

Experiments

Human breast cancer ZR75-1 cells were routinely cultured in Dulbecco’s Modified Eagle’s Medium containing 10% fetal calf serum and 1 O-’ M estradiol, as previously described (12). The transfected DNA included 5 fig reporter plasmid pBLCAT2 (36) containing ARE oligonucleotides at the Xbal site, 2 pg internal control plasmid, and pJ3 luciferase and pJ3 (37) to a total of 10 fig/dish. After transfection, cells were maintained in the absence or presence of 2.5 x 1 OF8 M dihydrotestosterone and harvested after 48 h. Cell-free extracts were assayed for CAT activity (38) and luciferase (39) to correct for differences in transfection efficiency. Acknowledgments We would like to thank Drs. De Vos and Verhoeven and their colleagues for their gift of the AR fusion protein, and I. Goldsmith for preparing the oligonucleotides used in this study. We would also like to thank Roy Pollock and Roger White for advice, and Mike Owen and members of the Molecular Endocrinology Laboratory for their comments on the manuscript.

Received July 23, 1992. Revision received September 28, 1992. Accepted September 28, 1992. Address requests for reprints to: Dr. Malcolm Parker, Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London, United Kingdom WC2A 3PX. * Permanent address: Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Melbourne, Parkville 3052, Australia. Supported by a block grant to the Howard Florey Institute of Experimental Physiology and Medicine from the National Health and Medical Research Council of Australia.

REFERENCES

1. Evans RM 1988 The steroid and thyroid hormone receptor superfamily. Science 240:889-895 2. Beato M 1989 Gene regulation by steroid hormones. Cell 561335-344 3. Ham J, Parker MG 1989 Regulation of gene expression by nuclear hormone receptors. Curr Opin Cell Biol 1:503511 4. Green S, Kumar V, Theulaz I, Wahli W, Chambon P 1988 The N-terminal DNA bindinq ‘zinc finqer’ of the oestroqen and glucocorticoid receptbrs determines target gene soecificitv. EMBO J 7:3037-3044 P 1989 Three 5. Mader S,‘Kumar V, de Verneuil H, Chambon amino acids of the oestrogen receptor are essential to its

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AR-Binding

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20

21

22.

Sequence

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