Identification of a Functional Androgen-Response Element in the Exon 1-Coding Sequence of the Cystatin-Related Protein Gene crp2

A. Devos, F. Claessens, P. Alen, J. Winderickx, W. Heyns, W. Rombauts and B. Peeters

Division of Biochemistry (A.D., F.C., P.A., W.R., B.P.) and Laboratory for Experimental Medicine and Endocrinology (J.W., W.H.) Faculty of Medicine University of Leuven B-3000 Leuven, Belgium


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Two hormone-responsive segments, one in the region of the promoter and one in intron 1, are identified in two homologous androgen-regulated and differentially expressed rat genes encoding the cystatin-related proteins (CRPs). Footprint analysis with the androgen receptor (AR) DNA-binding domain on the promoter-containing fragments reveals an AR-binding site downstream of the transcription start point in the crp2 gene (ARBSd/crp2, +40/+63). It displays an androgen response element-like sequence motif 5'-AGAAGAaaaTGTACA-3' and overlaps with the ATG translation start codon. A double-stranded oligonucleotide containing this sequence forms a DNA-protein complex with the full-length AR synthesized by vaccinia, as seen in band shift assays. Additional AR-binding sites, ARBSu/crp1 and ARBSu/crp2, occur 5' upstream of the transcription start point and are located at an identical position (-142/-120) in crp1 and crp2. The AR affinity for these two slightly different sequence motifs is relatively weak. The biological function of all three AR-binding sites as transcription control elements has been studied. The ARBSd/crp2 element clearly shows androgen-response element characteristics. The contribution of the common upstream element to the androgen-dependent control of reporter gene transcription is less clear. The transcription of a reporter gene construct containing the crp2 footprint fragment crp2F (-273/+88) is hormonally regulated as determined by transfection into the human breast cancer cell line T-47D. Androgens, but also glucocorticoids, efficiently stimulate steroid-dependent transcription of the chloramphenicol acetyltransferase gene. Mutation of the 5'-TGTACA-3' sequence in ARBSd/crp2 destroys the AR binding and abolishes the androgen-dependent synthesis of chloramphenicol acetyltransferase. A large fragment derived from intron 1 of the crp1 and crp2 gene can also provide the androgen-dependent transcription of chimeric constructs in T-47D cells. However, the induction measured is less than the one observed with crp2F (-273/+88), and this activity seems to reside in several subfragments that each display a low but consistent androgen responsiveness.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Steroid hormone receptors (SR) are intracellular receptors that act upon binding of their ligand to regulate gene transcription. Upon activation by their cognate hormone, the SRs form homodimers that are able to bind to an imperfect palindromic DNA sequence, called the hormone response element (HRE) (1). The receptors for glucocorticoids, mineralocorticoids, progestins, and androgens recognize the same DNA consensus sequence, which consists of an inverted repeat of two 6-bp half-sites, separated by a three-nucleotide (nt) spacer 5'-GGTACAnnnTGTYCT-3' (2). For the SRs, amino acid identity of the conserved DNA-binding domain (DBD) can be as high as 79% (3). In the context of this high homology in DBDs and HREs, it remains poorly understood how the SRs can achieve the steroid-selectivity and target gene specificity in vivo (4, 5, 6).

The cystatin-related protein genes (crps) are interesting candidates to study the mechanisms by which androgen-dependent gene expression and tissue specificity are obtained. CRPs are abundant glycoproteins almost exclusively synthesized in ventral prostate and lacrimal gland of the male rat (7). They are encoded by a multigene family (8) of which we have isolated two 90% homologous genes, crp1 and crp2 (9). A 20-kDa protein gene, identical to crp1, has been described by Ho et al. (5). In spite of the high homology between crp1 and crp2, their transcription is known to be differentially influenced by androgens in vivo. The crp1 response to androgens is fast and crp1 mRNA is detected in both ventral prostate and lacrimal gland. It can also be induced in female rats by androgen administration (10). The crp2 mRNA is exclusively detected in the ventral prostate, and its transcription is less sensitive to androgens (7).

The following report describes the search for functional AREs in these androgen-regulated crp genes. Up till now, these regulatory elements have mainly been found in the promoter regions for androgen-controlled genes such as those encoding prostate-specific antigen (PSA), sex-limited protein (Slp), probasin (PB), and mouse vas deferens protein (MVDP) (11, 12, 13, 14, 15). However, functional AREs have been detected in the first intron of the C3(1)-gene (16, 17) and the ninth intron of the ß-glucuronidase (GUS)-gene (18).

Chimeric constructs, containing fragments of a large region encompassing the 5'-upstream and intron 1 sequences of both crp genes, have been examined in the androgen-sensitive human breast cancer cell line T-47D. While fragments from intron 1 confer a weak to moderate androgen responsiveness to a reporter gene after transient transfection of chimeric constructs in these cells, a more significant response was observed with fragments originating from the transcription start point-containing region. A detailed analysis of these latter fragments has been made.

The deoxyribonuclease I (DNaseI) footprinting technique was used to locate AR-binding sites using a recombinant androgen receptor DNA-binding domain (AR-DBD) (19). A gene-specific footprint was detected in the coding sequence of the first exon of crp2 and a common footprint for crp1 and crp2 in their 5'-upstream regions. The protected sequences themselves were tested by gel retardation for AR-DNA complex formation and by mutational analysis to determine their role in the androgen-dependent response.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Selective Androgen Response of Two crp-mRNAs
Two highly homologous crp cDNA clones have been isolated from a library of rat ventral prostate mRNA (7). An androgen-dependent synthesis of the corresponding crp-mRNAs was demonstrated by selective hybridization with specific oligonucleotide probes. crp1-mRNA displays a fast response to changes in androgen concentration whereas its effect on crp2-mRNA is less pronounced as shown by mRNA dot-blot analysis (Fig. 1Go). To understand the difference in androgen responsiveness of these two crp genes, which display a 90% sequence identity (9), we analyzed promoter and intron regions to find the potential AREs involved in this process.



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Figure 1. Hormonal Regulation of mRNA Levels of crp1 and crp2

Total RNA was prepared from ventral prostates (five rats per sample) of intact rats and 1-, 3- and 7-days castrated animals and of 7-days castrates that were subsequently treated with androgens for 1, 3, or 7 days. Changes in mRNA concentrations were evaluated by dot-blot analysis, using the crp-specific oligonucleotides for crp1-mRNA (open squares) and crp2-mRNA (open diamonds) and densitometric scanning of the autoradiograms. The mRNA levels (estimated per milligram RNA) are expressed as percentages of the levels measured in intact male rats.

 
Androgen-Responsive Fragments in the crp Genes
To establish the endogenous crp promoter activity, the fragments (nt -584 to +11 for crp1 and nt -1389 to +46 for crp2) have been cloned in front of a chloramphenicol acetyltransferase (CAT) reporter gene (see Materials and Methods). Transient transfections were performed in T-47D cells and LNCaP cells, which both have a functional AR. In neither of these cell lines was measurable CAT activity obtained for the endogenous crp promoter constructs in the absence or presence of androgens (results not shown). Under the same conditions, the mouse mammary tumor virus (MMTV)-CAT construct and the C3(1)-intronic-fragment-thymidine kinase (TK)-CAT construct, containing core II sequence, the first functional ARE described in a cellular gene (16), and both used as positive controls, displayed, respectively, a 40- to 50-fold and a 2- to 3-fold steroid-induced transcription in the presence of androgens (10-6 M 5{alpha}-DHT or 10-9 M R1881).

As this transcriptional inactivity could be the result of cell type-specific promoter functioning or the presence of a silencer in the crp gene, a different approach was followed. The same promoter fragments, but also several subfragments generated from this region (see Materials and Methods), were tested in chimeric constructs, containing the TK-CAT reporter gene. The TK-promoter can activate gene transcription in T-47D and LNCaP cell lines (13, 16, 20). Under these conditions, the chimeric constructs can still be tested for the property of the inserted fragment to confer androgen responsiveness to the heterologous promoter. Transient transfections in T-47D cells revealed that a consistent 2- to 3-fold stimulated CAT activity can be obtained in the presence of 10-8 M 5{alpha}DHT or 10-9 M R1881 but only with vectors containing the crp1 Fragment (crp1F; nt -207 to +89) and crp2 Fragment (crp2F; nt -273 to +88) (Fig. 2Go). Several constructs, containing other regions of the 5'-upstream sequences (for crp1 nt -584 to +11 and crp2 nt -1392 to +46 see Fig. 2Go; crp1 promoter fragment nt -584 to -207 and crp2 promoter fragments nt -1037 to -586 and nt -585 to -273, results not shown), did not induce an androgen-dependent transcription.



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Figure 2. Analysis of Androgen-Dependent Enhancer Activities of Genomic Fragments Derived from Promoter and Intron 1 Region of crp1 and crp2 Genes

Panel A shows the overall structure of the crp1 and crp2 genes. The exons are represented by numbered boxes 1 to 4. The promoter and intron 1 region from which the crp-TK-CAT chimeric constructs are derived have been underlined (thick lines). In the schematic representation of the constructs, the lines represent the crp1- (panel B) or crp2- (panel C) derived fragments. Their location with respect to the transcription start point in the respective genes is indicated by the nucleotide number (9 ). The chimeric TK-CAT constructs were transiently transfected in T-47D cells that were grown in the absence or presence of the androgen R1881 (10-9 M). Transcription of the reporter gene was evaluated by measuring CAT activity as described in Materials and Methods. Experiments in which the human AR expression plasmid pSVAR0 was cotransfected are indicated (+). The relative induction factors were calculated as the absolute CAT activity of the constructs in the presence of hormone over the absolute CAT activity of the constructs in the absence of hormone. They are expressed as the -fold induction which is the mean value and SD of at least three independent experiments. No androgen inducibility was detected for the TK-CAT control vector (1.0 ± 0.1).

 
Large fragments derived from intron 1 of the crp1 and crp2 genes were cloned in front of TK-CAT and examined for androgen responsiveness. These 583- bp (crp1, nt 2274–2857) and 944-bp (crp2, nt 1955–2897) long fragments induced a 2-fold increase in CAT activity when transfected as chimeric constructs in T-47D cells and treated with 10-9 M R1881 (Fig. 2Go). Smaller restriction fragments obtained from the same intronic region and corresponding to the segments D1 (nt 2634–2857 for crp1, nt 2654–2897 for crp2) and D2 (nt 2503–2633 for crp1 and nt 2525–2653 for crp2) described and tested by Ho et al. (5), displayed equal or less androgen-dependent transcriptional induction (Fig. 2Go).

We reexamined all chimeric constructs in T-47D cells after cotransfection with pSVAR0, an expression plasmid for the human AR. These conditions explicitely demonstrate the involvement of the AR in the androgen-dependent transcription of the crp constructs. Essentially the same fragments are responsive to androgens but, depending on the constructs tested, their hormone-dependent transcription is slightly to highly increased (Fig. 2Go). While almost without effect on the control constructs, the level of androgen-induced CAT-synthesis for the crp2F (-273/+88) construct increased substantially from 3.1- to 13.6-fold. Such an increase was not observed for the crp1F (-207/+89) construct (Fig. 2Go). Both steroid hormones, 10-8 M 5{alpha}DHT and 10-9 M R1881, produce identical results.

The androgen-dependent transcription of some constructs containing large intron 1-derived fragments (nt 2274–2857 for crp1, nt 1955–2897 for crp2) is also increased after cotransfection of an AR expression vector (Fig. 2Go). Further analysis showed that the androgen responsiveness could not be traced back to a single small segment as both the D1 (2634/2857 for crp1, 2654/2897 for crp2) and D2 (2503/2633 for crp1 and 2525/2653 for crp2) subfragments displayed a moderate 2-fold induction by the hormone.

To find out whether the steroid response of the constructs containing the crpFs or intron 1 fragments is androgen specific, we tested the effect of a series of steroids in T-47D cells without SR cotransfection. The androgens 5{alpha}-DHT (10-5 to 10-10 M), testosterone (10-6 and 10-7 M), and R1881 (10-9 M) are all able to induce a 2- to 3-fold induction of the crpF-TK-CAT constructs. A similar level of induction is observed for the synthetic progestin R5020 (10-9 M) and for dexamethasone (10-6 M). The crp1F (-207/+89)-TK-CAT and crp2F (-273/+88)-TK-CAT constructs, cotransfected with either an expression plasmid for the human glucocorticoid receptor (hGR) or human androgen receptor (hAR) also revealed similar steroid hormone inducibility in COS1 cells treated with the corresponding hormone. CAT transcription, mediated by the different intron 1-containing constructs (Fig. 2Go), can also be efficiently stimulated by adding 10-6 M dexamethasone to the T-47D cells cotransfected with pRSVhGR (results not shown). The induction factors are similar to those observed for the same constructs, cotransfected with pSVAR0 in T-47D cells and treated with 10-9 M R1881.

Localization of AR-DBD Binding Sites by DNaseI Footprint Analysis
As the small crp2F (-273/+88) fragment confers a strong and consistent androgen inducibility to the CAT gene, we have focused on this fragment and the corresponding region of the crp1 gene. DNaseI footprinting was performed on both homologous fragments, using the purified recombinant AR-DBD fusion protein (19).

In crp2F (-273/+88) an AR-binding site ARBSd/crp2, displaying an ARE-like motif, is detected downstream of the transcription start point (nt +40 to +63) (Fig. 3AGo). The start codon of the CRP2 protein is included in this ARE-like sequence. The size of the protected region (24 nt) furthermore indicates that the AR-DBD molecules interact as dimers with the DNA. A clear hypersensitive site appears at the upstream boundary of the protected region (Fig. 3AGo, lane 2) In the corresponding crp1 sequence, which displays one A to G base exchange and the insertion of an A, no footprint was detected (Fig. 3BGo, lane 2).



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Figure 3. Analysis by DNaseI Footprinting of AR-DBD Interaction with Sequence Motifs in the Promoter Fragments of crp1 and crp2

Fragments from crp2, crp2F (nt -273 to +88), and its mutated form crp2F/ARBSm (panel A), and from crp1 and crp1F (nt -207 to +89; panel B) are labeled at the 3'-end of the sense strand. In panels C and D, the corresponding fragments are labeled at the 3'-end of the antisense strand. The radioactive probes were incubated with or without AR-DBD, AR-DBD + competitor, or protein A and digested with DNaseI (see below). The sequences and positions in crp1F and crp2F protected from nuclease attack are depicted at the left of each window, and the hypersensitive site is indicated by an arrowhead. At the right, sequences are given of the region displaying no interaction with AR. Base differences between crp2F and crp1F in this region are marked by an asterisk. The DNaseI-treated probes have been aligned with the G (or G+A) and A+C chemical cleavage products of the corresponding fragment. A, crp2F is incubated without (lane 1) or with 0.6 pmol AR-DBD (lane 2–6). crp2F/ARBSdm is incubated without (lane 7) or with 3 pmol AR-DBD (lane 8). Competition is with a 100-fold excess of ds oligonucleotide containing core II (lane 3), a 400-fold excess of ds oligonucleotide containing ARBSd/crp2 (lane 4), a 1000-fold excess of ds oligonucleotide containing ARBSdm/crp2 (lane 5), and a sequence containing the NF1-Ad-binding site (lane 6). B, The crp1F is incubated without (lane 1) or with a 0.6 pmol AR-DBD (lane 2). C, The crp2F is incubated without (lane 1) or with 1.8 pmol AR-DBD (lane 2–5). Competition is with a 100-fold excess of ds oligonucleotide containing core II (lane 3), a ds oligonucleotide containing ARBSu/crp2 (lane 4), and a 1000-fold excess of ds oligonucleotide containing NF1-Ad-binding site (lane 5). D, The crp1F is incubated without (lane 1), with 1.8 pmol AR-DBD (lanes 2–3), and with 3.6 pmol protein A (lane 4). Competition is done with a 100-fold excess of ds oligonucleotide containing core II (lane 3).

 
Competition experiments on ARBSd/crp2 with a 100-fold molar excess of a double stranded (ds) oligonucleotide representing the core II ARE (16) resulted in a complete loss of the AR-DBD-derived footprint (Fig. 3AGo, lane 3). Instead, a 400-fold excess of unlabeled ds oligonucleotide, containing the sequence motif of the protected region itself, was required to displace the AR-DBD dimer from the labeled fragment (Fig. 3AGo, lane 4). Moreover, a ds oligonucleotide encompassing the ARBSdm/crp2, in which the 5'-TGTACA-3' has been mutated to a PvuII restriction site, or a ds oligonucleotide encoding the NFI-Ad motif, was unable to interfere with the DNA-protein complex formation even at a 1000-fold excess (Fig. 3AGo, lanes 5 and 6). Conversely, when the mutated crp2F (-273/+88) was used in DNaseI footprinting, no window was observed, even when the AR-DBD was added at 3-fold higher concentration (Fig. 3AGo, lane 8).

A second, but fainter AR-binding site ARBSu/crp2 (nt -136 to -115) was observed in the 5'-upstream region on crp2F (-273/+88) (Fig. 3CGo, lane 2). The corresponding region in crp1F (-207/+89) was also protected by AR-DBD against DNaseI digestion, resulting in a footprint ARBSu/crp1 (nt -142 to -121) (Fig. 3DGo, lane 2). These footprints are located at identical positions in both genes as can be deduced from the sequence alignment for optimal sequence identity by the PC Gene program (9). The protected sequences mutually differ in two nucleotides. Protein A, to which the AR-DBD is linked, is not responsible for the footprint as it does not confer protection of the ARBSu/crp1 against DNaseI (Fig. 3DGo, lane 4). Here too, mutation of the 3'-half-site 5'-TGTACT-3' sequence of the putative AREs to a PvuII restriction site abolishes protection, even when the AR-DBD is added at 5-fold higher concentration (results not shown).

Full-Length AR Binds Differentially to the crp-ARBS Sequences
To compare the affinity of the AR for the binding motifs observed in footprint analysis, gel retardation experiments were performed with labeled ds oligonucleotides encompassing the complete protected sequences. For this analysis, a full-size AR overexpressed in HeLa cells by the vaccinia system was used instead of the AR-DBD recombinant protein. Although the latter protein produces a stable protein-DNA complex with the core II motif taken from the C3(1) gene intron, no complexes were retarded using oligonucleotides containing the ARBS-derived sequences (6). The full-length AR interacts with core II (Fig. 4AGo, lane 3–6) but not with its mutated form, even when AR-Ab are added (Fig. 4AGo, lane 8–9) because only weak bands of nonspecific complexes also present in HeLa nuclear extract (Fig. 4AGo, lane 1) are seen. However, with the full-length AR, not only complex formation was observed with the core II sequence, but also with the oligonucleotides containing ARBSd/crp2 and even with the ARBSu/crp2 and ARBSu/crp1 sequences. Using an equal labeling specificity of the different probes, the detection of the DNA/AR complex requires an almost 6-fold longer exposure time (Fig. 4BGo). The complex formation could be improved by the addition of AR antibodies, especially with the ds probes containing sequence motifs of lower receptor binding affinity, and resulted in a supershift of the specific AR-DNA complex. Furthermore, competition experiments revealed the specificity of these interactions. The AR-ARBSd/crp2 complex was absent after addition of a 200-fold excess of the unlabeled ds oligonucleotide (Fig. 4BGo, lane 8). Although at this competitor concentration, no effect was seen on the amount of AR-core II complex formed (Fig. 4BGo, lane 3), this competition can be observed when the film is not overexposed (Fig. 4AGo, lane 4). This indicates that the receptor has a lower affinity for this competitor sequence than for the core II motif, which displays the highest homology with the consensus GRE/ARE.



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Figure 4. Analysis by EMSA of AR Binding to DNA Probes Containing Putative ARE-Like Sequence Motifs Occurring in the Promoter Regions of the crp Genes

A, 104 cpm of labeled ds oligonucleotides containing sequence motifs of core II (lanes 1–5) or a mutated core II (lanes 6–9) were incubated with pure HeLa extract (lane 1) or the nuclear extract containing full-length AR synthesized in vitro by the vaccinia system. AR-DNA complex formation is analyzed by gel retardation assay as described in Materials and Methods. The open circle indicates the aspecific bands. The black arrowhead indicates the position of the AR-DNA complex. The location of a supershifted complex obtained in the presence of AR antibodies is indicated by an open arrowhead. In the competition experiment (lane 4), a 200-fold molar excess of unlabeled ARBSd/crp2 oligonucleotide is added. B, 104 cpm of labeled ds oligonucleotides containing sequence motifs of core II (lane 1–4), ARBSd/crp2 (lane 5–9), ARBSu/crp1 (lane 10–12), and ARBSu/crp2 (lane 13–15) were incubated with full-length AR synthesized in vitro by the vaccinia system. AR-DNA complex formation is analyzed by gel retardation assay as described in Materials and Methods. The black arrowhead indicates the position of the AR-DNA complex. The location of a supershifted complex obtained in the presence of AR antibodies is indicated by an open arrowhead. In competition experiments, a 100- to 200-fold molar excess of unlabeled ARBSd/crp2 oligonucleotide is added as indicated in the different lanes.

 
Effect of Mutations of the crp-AREs on the Response to Androgens
To investigate whether the AR recognition motifs in the footprints are indeed responsible for the androgen-dependent transcriptional expression of the crp2F (-273/+88)-TK-CAT construct, the 5'-TGTACA-3' sequence for ARBSd/crp2 and the 5'-TGTTCA-3' sequence for ARBSu/crp2 have been mutated to a 5'-CAGCTG-3' PvuII restriction site (crp2F (-273/+88)/ARBSdm and crp2F (-273/+88)/ARBSum). This modification does not change the overall configuration of the construct but, as already shown, completely abolishes the AR-DBD binding in DNaseI footprinting (Fig. 3Go). The mutated fragments were cloned in front of TK-CAT in analogy to their nonmutated counterparts. As shown in Fig. 5Go, crp2F (-273/+88)/ARBSdm, which carries the mutation in ARBSd/crp2, is no longer able to confer androgen responsiveness to the TK promoter when cotransfected with pSVAR0 in T-47D cells supplied with androgens. On the other hand, the construct containing ARBSum/crp2 is still able to mediate androgen-dependent gene transcription at a slightly lower level than the original construct. Remarkably, the corresponding AR-binding site (ARBSu/crp1) in the crp1 gene was most probably responsible for the 17-fold transcriptional induction by androgens observed with the crp1F (-207/+89)-TK-CAT construct tested under identical conditions in one specific T-47D lineage that is no longer available for study. Indeed, mutation of the 3'-half-site of the AR-DBD interacting motif to a PvuII restriction site in this single element of the construct led to the complete absence of androgen-dependent CAT expression (results not shown).



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Figure 5. Functional Analysis of AR-Binding Sites in crp2F (-273/+88) Fragment

The crp2F (-273/+88)-TK-CAT construct and its derivatives are transiently cotransfected with pSVAR0 in T-47D cells. Cells were grown in the absence or presence of hormone R1881 (10-9 M), and transcription of the reporter gene is measured by CAT assay as described in Materials and Methods. The schematic representation of the different constructs represent the crp2F segment linked to TK-CAT. In crp2F, the transcription start point is indicated by a dashed arrow, and the 5'- and 3'-ends by their nucleotide number (9 ). The location of the receptor-binding sites, ARBSu/crp2 (O) and ARBSd/crp2 (@) is indicated by the nucleotide number and the mutated motifs are presented by (X). The position of the restriction site to generate subfragments is also marked by the nucleotide number. The relative induction factors were calculated as the absolute CAT activity of the constructs in the presence of hormone over the absolute CAT activity of the constructs in the absence of hormone. Results are presented as an n-fold induction, which is the mean value and SD obtained of at least three independent transfection experiments. No androgen inducibility was detected for the TK-CAT control vector (1.0 ± 0.1).

 
The involvement of one or both AR-binding sites in the hormone response of the crp2F (-273/+88)-containing construct in transfections has subsequently been examined by testing two different constructs, containing either the 5'-upstream part (crp2Fu, nt -273 to -98) or the 3'-downstream located part (crp2Fd, nt -98 to +88) of this region and therefore only a single receptor interacting motif. As indicated in Fig. 5Go, only the fragment containing the ARBSd/crp2 sequence conferred androgen-mediated transcriptional activation. The induction values observed are similar to those measured for the full-length crp2F (-273/+88) construct, thereby substantiating the results seen in the previous experiments with the crp2F (-273/+88) mutants.

Finally, we could demonstrate that ARBSd/crp2 is an enhancer sequence. Indeed, inserted in the opposite orientation crp2(F-) (+88/-273) in front of the promoter, thereby increasing the distance between ARBSd/crp2 and other regulatory elements in the TK-promoter from 38 bp to 220 bp, it is still able to induce a moderate androgen-dependent gene transcription (Fig. 5Go).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The expression of crp1, also described as the 20-kDa protein gene (5), and crp2 (9) is androgen-dependent in vivo (Fig. 1Go). In this study, gene fragments of these two 90% homologous crp genes have been evaluated for their ability to confer androgen responsiveness on a reporter gene in transient transfection experiments.

As no CRP-synthesizing epithelial cell lines are available, T-47D cells have been used. They contain a functional AR, and androgen responsiveness of several fragments from other steroid-controlled genes has been successfully investigated in this cell line (13, 16, 21). To take into account the possibility of cell type-specific gene expression (22), the hormone-dependent transcription of some chimeric constructs has also been tested in LNCaP and COS1 cells.

Putative ARE-like sequences were detected in the 5'-upstream regions of the crp genes (9), but identification of these elements by their functional analysis in the context of the endogeneous crp promoter has been unsuccessful. Similar observations of promoter inactivity have been made for constructs of the 20-kDa protein gene transfected into CV1 cells (5) and of the C3(1) gene when tested in T-47D and CV1 cells (16, 17).

Using the viral TK-promoter, which is functional in a wide range of different cell lines, a low but consistent androgen-induced transcription of CAT was seen in transiently transfected T-47D cells for the crpF-TK-CAT (-207/+89 for crp1 and -273/+88 for crp2) constructs and those containing intron 1-derived fragments (Fig. 2Go). Cotransfection of pSVAR0 in T-47D cells did improve the androgen-dependent transcription of some of these constructs. Unexpectedly, the induction level of the crpF containing TK-constructs depends on the batches of T-47D cells taken for transfection.

Three successive samples of T-47D cells, obtained from ATCC (Rockville, MD), display a clear R1881-dependent transcription of crp2F (-273/+88)-TK-CAT. However, the highest transcriptional enhancement by R1881 (17-fold induction) has been observed with the crp1F(-207/+89)-TK-CAT construct. This induction level has been consistently seen in a large number of transfection experiments but only in one specific, fast proliferating T-47D cell batch, which displays an insulin-independent growth.

Our transfection results of crp1 and the observations made by Ho et al. (5) revealed a cell-specific androgen response. In T-47D cells, we did not find any androgen responsiveness for the 5'-upstream gene fragment (-584/+11) whereas these authors have detected a 2-fold induction of CAT transcription by androgens when a fragment (-582/-182) from this region was tested in CV1 cells. This discrepancy could be due to the presence of cell type-specific silencer elements that affect the androgen-dependent transcription as has been shown for some other genes (12, 13, 14).

For constructs containing intron 1 fragments and tested in T-47D cells, the level of androgen inducibility is always lower than the one observed with the crpF constructs. In contrast, similar intron 1 fragments reveal a higher androgen responsiveness in comparison with the constructs containing the promoter region when examined in CV1 cells (5). In addition, the hormone selectivity of the intron fragments observed by Ho et al. (5) in CV1 cells has not been seen in T-47D cells. This lack of hormone-specific response has also been observed during promoter studies of the MVDP gene in the T-47D cell line (15) and of the PSA gene in the homologous cell line LNCaP (20).

DNaseI footprinting on the crpF fragments with a recombinant fusion protein containing the AR-DBD (19) reveals two windows in crp2F (-273/+88) and one in crp1F (-207/+89). The region shielded from DNaseI attack indicates that the AR-DBD interacts as a dimer, as its size is comparable to the one previously observed on the core II motif in the intron 1 fragment of the C3(1) gene and on the distal HRE of the long terminal repeat region in MMTV (23).

All three AR-binding motifs differ at position -2 of the highly conserved consensus sequence for an ARE (5'-AGAACAnnnTGTTCT-3') (2) as deduced from functional elements identified in a number of androgen-regulated genes (15). This base exchange strongly affects the stability of the DNA-receptor complex (24, 25). This has also been demonstrated in footprinting analysis on the crpFs by competition experiments with either the core II motif of C3(1) or the ARBS/crp derived probes. Moreover, mobility shift analyses using the AR-DBD only reveal DNA-protein complex formation with the core II ds oligonucleotides, and not with the sequences containing ARBS/crp motifs. However, the full-size AR interacts both with the core II- and ARBSd/crp2-sequences. ARBSu/crp1- and ARBSu/crp2-receptor complexes could only be clearly detected after stabilization with AR-antibodies, indicating AR interaction with gene fragments displaying weak affinity binding sites (5, 14).

The ARBSd/crp2 contains an ARE-like motif that very well resembles the proximal ARE (5'-TGAAGTtctTGTTCT-3') described in the MVDP gene promoter (15). The corresponding region in the crp1 gene shows no AR-DBD interaction. This is most probably due to the insertion of an A in the spacer region between the two 6-bp half-sites of the binding motif. The 4-bp spacing completely destroys binding of SRs to their recognition site as has been clearly demonstrated for the GR and PR (6, 24, 25).

The role of these AR-binding sites, ARBSd/crp2, ARBSu/crp2, and ARBSu/crp1 showing a moderate to weak affinity for the receptor, has been identified in the crpFs. ARBSd/crp2 consistently confers androgen response to the heterologous promoter in T-47D cells cotransfected with an hAR expression plasmid and supplemented with R1881. The location of the ARBSd/crp2 in the coding region of exon 1 is most unusual, as AREs identified downstream of the transcription start point have thus far been found in introns (16, 18). Its function as enhancer has been confirmed by the fact that crp2(F-) (+88/-273)-TK-CAT is still able to confer androgen-dependent induction. The decrease in induction level is most probably the result of a positional effect. Increasing the intervening sequence between a HRE and other regulatory elements displaying cooperative interaction through their bound transcription factors can indeed affect the steroid-dependent transcription of a reporter gene (26, 27, 28). The mutation of the 3'-half-site of ARBSd/crp2, which does not change the overall configuration of crp2F (-273/+88)-TK-CAT, completely abolishes its androgen-dependent transcription.

The role as an ARE of the common ARBSu/crp motifs is less clear. The ARBSu/crp2 does not seem to participate in controlling the androgen response of the crp2F (-273/+88) chimeric construct in the T-47D clones that we are currently using. Mutation of ARBSu/crp2 hardly affects the androgen-dependent transcription of the mutated construct (Fig. 5Go). These results are substantiated by the observation that another construct containing only the 3' half of crp2F (-98/+88), and thus only the ARBSd/crp2 motif, displays almost the same level of induction as the complete fragment. Nevertheless, in a series of experiments using a fast proliferating, insulin-independent T-47D clone, we observed that destroying the AR binding to ARBSu/crp2 had an effect on the androgen responsiveness. The crp2F/ARBSum construct showed a significant drop (40%) in steroid-dependent transcription (results not shown). These results could indicate that a functional interaction may exist between the two ARE-like elements. Similar observations of cooperative interaction between AREs have been described for the probasin gene promoter (14). A further proof that AR recognition motifs displaying weak affinity for the receptor can still function as strong AREs in transfection experiments, when positioned in their appropriate DNA context and transfected in a responsive T-47D clone, is demonstrated for the crp1F (-207/+89) construct. This fragment, containing a single AR-binding site (ARBSu/crp1) with a sequence and receptor binding property almost identical to the ARBSu/crp2 motif, can confer a 17-fold induced transcription by R1881. The fact that a small mutation in the 3'-half-site of the ARBSu/crp1 sequence, destroying the AR binding, can completely shut off the androgen-dependent transcription of the reporter gene clearly underlines its function as an ARE. In vitro binding experiments reveal that the ARBSu/crp1 regulatory element is part of a complex androgen response unit. DNaseI footprinting on the crp1F (-207/+89), using nuclear extracts of rat ventral prostate and lacrimal gland tissue, has shown that an array of binding sites for different transcription factors is flanking this ARBSu/crp1 site and the pattern of footprints produced is distinct for the crp1 and crp2 promoter (unpublished results).

The fact that the highly homologous segments crp1F and crp2F confer a quite different hormone responsiveness when tested as chimeric constructs in transient transfections may provide some potential clues to understanding the differential response of the two genes as seen in vivo. Moreover, functional AREs have been identified in these androgen-responsive crp promoter fragments. They differ in number and affinity as demonstrated by in vitro binding studies, and the sequences are markedly different from the consensus ARE. Furthermore, the location of the ARBSd/crp2 motif is of particular interest. Indeed, while most AREs detected up till now are found in the 5'-upstream and intron regions, the ARBSd/crp2 is located in exon 1 over the translation start site of CRP2, and therefore it is the first ARE described in a coding sequence of a gene.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Messenger RNA Preparation and Dot-Blot Hybridization Assays
Male Wistar albino rats (3 months old) were used for this study. Castration was performed under ether anesthesia. Some castrated animals received subcutaneous injections of androgens (0.5 mg testosterone propionate and 0.5 mg testosterone in 0.2 mg olive oil). Before removal of organs, rats were anesthetized with ether and bled to death via the carotid artery. Immediately after killing, organs were removed and frozen in liquid nitrogen. Dot-blot analysis on total RNA from ventral prostates was performed as described by Winderickx et al. (7). The crp-specific oligonucleotides, 5'-ACCCTACATGGCACACTGCT-3' (crp1-mRNA) and 5'-ACCCTATGTGGCACACAGCT-3' (crp2-mRNA) were used in hybridization at a temperature of 57 C. An 18S rRNA-specific oligonucleotide probe was hybridized at 72 C and used as a control (7). The mRNA levels (estimated per mg RNA) are expressed as percentages of the levels measured in intact male rats.

Construction of CAT Fusion Plasmids
The crp-gene subfragments were obtained either by restriction enzyme cleavage or PCR amplification and were cloned into the pBLCAT2 vector, containing a TK promoter linked to a CAT reporter gene (30). pBLCAT2 was digested with SalI and used as such or made blunt-ended by a fill-in reaction. Recombinant clones with specific inserts were selected, and vector/insert overlapping regions were analyzed by sequencing.

A BamHI-ScaI genomic crp1 fragment (nt -584 to +292) (9), containing the promoter region, was digested by HaeIII, and the purified subfragments of 379 bp and 296 bp were cloned (crp1PF1, -584/-207 and crp1F, -207/+89). For the crp2-promoter region, an AccI-ScaI fragment (nt -1037 to +291) was treated with HaeIII and fragments of 454 bp, 312 bp, and 358 bp were inserted in the vector (crp2PF1, -1037/-586; crp2PF2, -585/-273; crp2F -273/+88). From the 358-bp fragment that contains the transcription start point, two subfragments were generated by HinfI-digestion and subcloned (crp2Fu -273/-98 and crp2Fd -97/+88).

The crp1-promoter fragment (-584/+11) was obtained by serial deletion at the 3'-end using the Erase-a-base system (Promega, Madison, WI). Religated plasmids were transfected in XL1-Blue cells (Stratagene, La Jolla, CA), and clones containing the recombinant plasmids, displaying inserts of about 600 bp, were analyzed by sequencing to determine the exact 3'-end of the gene insert. After SalI/NsiI digestion, the promoter fragments were cloned in the SalI/PstI double-digested vectors pCATEnh vector (Promega) or pBLCAT2 (crp1P: -584/+11).

A fragment containing the complete 5'-upstream region of the crp2-gene (nt -1392 to +46), cloned in pGEM-7Zf(+), has been made by PCR (Gene Amp Reagent kit with amplitaq; Cetus Inc, Berkeley, CA) with the primers 5'-aaagtcgacagatctATCCAGAGTCCTGGG-3' (nt -1392/-1375) and 5'-aaagtcgacggatccTCAGATATGAAAGGG-3' (nt +30/+46). These primers were designed to contain restriction sites at their 5'-ends. All PCR reactions have been performed on plasmid DNA, prepared either by CsCl gradients or Qiagen columns. PCR reactions were carried out according to the protocols. After SalI digestion, the promoter fragments were cloned in the SalI-site of the pCATEnh vector (Promega) or pBLCAT2 (crp2P: -1392/+46).

Genomic fragments of intron 1 were derived from the 3445-bp crp1 BamHI fragment (nt -574 to +2857). After digestion with HinfI, a segment of 474 bp (nt 2274–2857) was selected. D1 and D2 fragments were obtained from HindIII digestion of the same BamHI crp1 fragment, followed by a DdeI digestion. The fragments of interest are crp1D1 (nt 2634–2857) and crp1D2 (nt 2503–2633). For crp2, a BamHI fragment (nt -1393 to +2897) was cleaved with Sau3AI yielding a fragment of 944 bp (nt 2955–2897). crp2D1 and crp2D2 were derived from the same 4287-bp BamHI fragment, digested by XbaI and subsequently digested by DdeI. The fragments crp2D1 (nt 2654–2897) and crp2D2 (nt 2525–2653) were subcloned in the pBLCAT2 SalI site, which had been made blunt-ended.

PCR-mediated mutagenesis was performed according to Higuchi et al. (31). For crp1, the oligonucleotides 5'-TGGGTGAGGGAACAAGCAGCTGATAGGGAATGAAGTT-3' (-145/-210) and 5'-AACTTCAATCACTATCAGCTGCTTGTTCCCTCACCCA-3' were used for the generation of the ARBSu/crp1 mutant. On crp2F, oligonucleotides 5'-TGGGTGAGGTAAAAAGCAGCTGATAGGGAATGAAGTT-3' (-239/-204) and 5'-AACTTCATTCCCTATCAGCTGCTTTTTACCTCACCCA-3' were taken to obtain the ARBSu/crp2 mutant and a combination of the oligonucleotides 5'-TATCTGAGAAGAAAACAGCTGAAACCCTATGTGGCA-3' (+37/+73) and 5'-TGCCACATAGGGTTTCAGCTGTTTTCTTCTCAGATA-3' results in the ARBSd/crp2 mutation. The PCR products were subcloned in a pCRScript vector (pCR-Script SK(+) Cloning kit, Stratagene). Inserts were tested for the presence of the required mutation by restriction analysis with PvuII before subcloning in pBLCAT2. For crp1, the clone carrying the mutation in ARBSu/crp1 (crp1F/ARBSum) was obtained; for crp2, crp2F/ARBSum carries the mutation in ARBSu/crp2; crp2F/ARBSdm has the mutation in ARBSd/crp2. All inserts produced by PCR were verified by complete sequencing of the inserts.

Cell Culture and Hormones
Three batches of the human breast cancer cell line T-47D were ordered at different occasions at the American Type Culture Collection (Rockville, MD). Batch 1 contained ±125 fmol AR, ±50 fmol GR, and ±480 fmol PR per mg protein. This company also provided the human lymph node carcinoma of prostate cell line LNCaP and the African green monkey kidney cell line COS1. T-47D cells were grown in DMEM containing 1000 mg/liter glucose, supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin, 4 µg/ml insulin, and 10% FCS (Sera-Lab, Sussex, England; or GIBCO-BRL, Gaithersburg, MD). LNCaP cells were grown in the same medium, supplemented with L-glutamine (2 mM). For COS1 cells, DMEM containing 4500 mg/liter glucose, 20 mM HEPES, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 5% FCS was used as growth medium. All cell lines were regularly checked for Mycoplasma contamination (Mycoplasma detection kit, Boehringer Mannheim, Mannheim, Germany).

T-47D and COS1 cells were transfected by the calcium phosphate coprecipitation transfection method (16). For cotransfections, 4.5 µg plasmid DNA were used to which 0.25 µg pCMV-LUC control vector and 0.25 µg pSVAR0 were added. The transfection method by lipofectin has been optimized for LNCaP cells, according to the accompanying protocols. pMMTV-CAT has been described by Cato et al. (32) and was used as a positive control on hormone response for each set of transfection experiments. To allow comparison of the transcription conferred by each of the promoters, the CAT activity was corrected for differences in transfection efficiency, as measured by the luciferase activity resulting from the cotransfected pCMV-LUC-vector.

The cells were maintained in the presence or absence of 10-5 to 10-10 M 5{alpha}-dihydrotestosterone (5{alpha}-DHT), 10-6 or 10-7 M testosterone, 10-9 M Methyltrienolone (R1881), 10-6 M dexamethasone, or 10-9 M promegestone (R5020). Culture medium with or without hormone was replaced every 24 h. Transfections were repeated at least three times, using at least two different plasmid preparations.

CAT and Luciferase Assays
Cells were harvested in 500 µl of 1x reporter lysis buffer (Luciferase cell culture 5x lysis buffer, Promega) and allowed to stand at room temperature for 15 min. Lysed cells were collected, transferred to Eppendorf tubes, vortexed for 1 min, placed on ice for 10 min, and again vortexed for 1 min. After the lysate was spun for 3 min at 13,000 rpm, supernatant was transferred to a new tube. Protein concentration of supernatant was determined with the Bradford reagent in microtiter plates (DC Protein Assay System, Bio-Rad, Richmond, CA). For corresponding samples with and without hormone, protein concentration was equalized. Luciferase activity was measured on 10 µl of lysate in a MicroLumat LB96P (EG&G Berthold, Badwildbad, Germany) using the luciferase assay kit (Promega) according to the protocol provided by the company.

Lysate (300 µl) was used to perform the CAT assay with acetyl coenzyme A (AcCoA) obtained from Pharmacia (Piscataway, Uppsala, Sweden) and [14C]chloramphenicol (CAM) from Amersham (Little Chalfont, England). The labeled CAM and derivates were extracted with ethylacetate (Janssen Chimica, Beerse, Belgium), spotted on TLC plates, and separated in CHCl3-MeOH (19:1). Radioactive spots were quantified by use of the PhosphorImager and the accompanying software (Molecular Dynamics, Sunnyvale, CA). Relative induction factors are expressed as the mean and SEM of at least three independent experiments.

Labeling DNaseI Footprint Fragments
The restriction fragments of crp1 (nt -207 to +89, footprint fragment crp1F) and of crp2 (nt -273 to +88, footprint fragment crp2F), subcloned in the EcoRI site of pGEM-7Zf(+), were used to prepare the end-labeled probes necessary in DNaseI footprinting.

For the upper strand, pGEM-7Zf(+) plasmids, containing the crp1F or crp2F, were digested with ClaI, labeled with Klenow DNA polymerase (Pharmacia) and [{alpha}-32P]dCTP (Amersham International) and redigested with SphI. For the lower strand, the first digestion was with XbaI, the ends filled in with [{alpha}-32P]dCTP, and the fragments were subsequently digested with SacI. The labeled DNA fragments were purified on a 4% nondenaturing polyacrylamide gel (acrylamide-bisacrylamide 29:1; 1x Tris-borate-EDTA) by electrophoresis for 2 h at 120 V. The bands were cut out of the gel after they had been localized by means of an x-ray film, and DNA was extracted from the gel fragments by electro-elution. DNA was precipitated with ethanol and redissolved at 10,000 cpm/µl in water.

DNaseI Footprinting Analysis
DNaseI footprinting experiments were performed according to Celis et al. (29). An almost pure fusion protein of the DBD (amino acids 540–607) of the AR, linked to protein A and expressed in Escherichia coli, was obtained by affinity chromatography on IgG Sepharose (19).

Labeled probe (30,000 cpm) was incubated with 600 fmol to 5 pmol AR-DBD fusion protein for 20 min at 20 C. For the competition experiments, the competitor DNA was also added at this stage. Digestion with 100–500 mU DNaseI (Boehringer Mannheim, Mannheim, Germany) was performed during 60 sec at 20 C in a final volume of 50 µl. The digested DNA probe was analyzed on a denaturing (7 M urea) 8% polyacrylamide sequencing gel.

The G and (A+C)-degradation reactions were performed, according to Maxam and Gilbert (33). For each footprinting experiment, the DNaseI-treated end-labeled probes are aligned with the sequence produced by this partially chemically cleaved probe to locate putative sites of interaction.

Oligonucleotides
Synthetic complementary oligonucleotides were used as ds probes that were labeled for electrophoretic mobility gel shift assay (EMSA). Unlabeled oligonucleotides were taken as competitor in the DNaseI footprinting and EMSA experiments. The core II containing oligonucleotide (5'-agcttACATAGTACGTGATGTTCTCAAGgtcga-3') has the ARE motif of the C3(1) intronic fragment. Its mutated counterpart carries a mutation in the 3'-half-site of this ARE motif (5'-agcttACATAGTACGTGATTTTCTCAAGgtcga-3'). NF1-Ad (5'-ATTTTGGCTACAAGCCAATATGAT-3') contains the binding site in adenovirus for nuclear factor 1. ARBSu/crp1 (5'-agcttGTGAGGGAACAAGTGTACTATAGgtcga-3'), ARBSu/crp2 (5'-agcttGTGAGGTAAAAAGTGTACTATAGgtcga-3'), and ARBSd/crp2 (5'-agcttTCTGAGAAGAAAATGTACAAAACCatcga-3') represent the sequence motifs protected by AR-DBD in DNaseI footprinting on the crppromoter fragments.

EMSA
EMSA was performed as described by De Vos et al. (34). The full-length AR has been prepared in a vaccinia system (35). [{alpha}-32P]dATP-labeled probe (20,000 cpm) was incubated for 20 min at 20 C in a final volume of 20 µl with 2.5–5 µg HeLa cell nuclear extract, either containing the AR (5–10 fmol AR) or not. The samples were separated on a 4% polyacrylamide gel (acrylamide-bisacrylamide, 29:1) in 0.25 x TBE-buffer. To some of the reaction mixtures, rabbit polyclonal antibody (Ab) against the N-terminal domain of the AR (AR-Ab) have been added. For the competition experiments in gel retardations, 100- or 200-fold excess of unlabeled probe was added to the incubation mixture.


    ACKNOWLEDGMENTS
 
The authors greatly appreciate the synthesis of oligonucleotides by V. Feytons and the excellent technical assistance of R. Bollen and H. De Bruyn. pCMV-LUC control vector was kindly provided by Dr. Nelles, Celgen, Katholicke Universiteit, Leuven; pSVAR0 was a gift from Prof. Dr. Brinkmann, and pRSVhGR was a gift of Prof. Dr. R. Evans.


    FOOTNOTES
 
Address requests for reprints to: Dr. B. Peeters, Faculteit Geneeskunde, Afdeling Biochemie, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium.

This work was supported in part by Grant ’Geconcerteerde Onderzoeksactie van de Vlaamse Gemeenschap’ and by Grant 3.0048.94 from the Belgian ’Fonds voor Geneeskundig Wetenschappelijk Onderzoek.’ A.P. is supported by a scholarship of the ’Vlaams Instituut voor de Bevordering van het Wetenschappelijk-Technologisch Onderzoek in de Industrie.’ C.F. is Senior assistant of the ’Nationaal Fonds voor Wetenschappelijk Onderzoek.’

Received for publication September 23, 1996. Revision received April 14, 1997. Accepted for publication April 17, 1997.


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 DISCUSSION
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