©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Protooncogene c- jun Contains an Unusual Estrogen-inducible Enhancer within the Coding Sequence (*)

Salman M. Hyder (1) (2)(§), Zafar Nawaz (3), Constance Chiappetta (1), Koshinaga Yokoyama (4), George M. Stancel (1)

From the (1) Departments of Pharmacology and (2) Obstetrics, Gynecology, and Reproductive Sciences, University of Texas Medical School, Houston, Texas 77225, the (3) Department of Cell Biology, Baylor College of Medicine, Houston, Texas, 77030 and the (4) Tsukuba Life Science Center, RIKEN, Ibaraki 305, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Estrogens have previously been shown to induce c- jun mRNA levels in target cells during hormone induced proliferation, and this appears to be a primary hormonal response involving transcriptional activation. In this report we have now identified an estrogen dependent enhancer within the coding sequence of c- jun. This element has the sequence GCAGA nnnTGACC which is identical to the consensus estrogen response element GGTCA nnnTGACC in the second half site, but varies considerably in the first half site. Synthetic oligodeoxynucleotides containing this jun sequence bind the estrogen receptor in cell-free studies using a competitive band shift assay with the consensus element. The jun element also confers hormone inducibility to reporter plasmids in yeast and mammalian based transcriptional systems. Structure-function studies illustrate that the TGACC half-site and its immediate flanking dinucleotides, but not the GCAGA half-site, are required for estrogen receptor binding. In contrast, both the GCAGA and TGACC half-sites are obligatory for hormone-inducible transcriptional activation. These results suggest a model in which the estrogen receptor functions as a heterodimer to regulate transcription of the c- jun protooncogene. Coupled with reports of estrogen response elements in c- fos and estrogenic induction of c- fos and c- jun in vivo, these findings also support a role for AP-1 components as early response genes in estrogen-induced proliferation.


INTRODUCTION

The estrogen receptor (ER)() belongs to a large family of related transcription factors that includes receptors for steroid hormones, vitamin D, thyroid hormone, retinoic acid, and many orphan receptors for which the ligands have not yet been identified (1, 2) . These receptors bind to specific nucleotide sequences in the DNA, known as hormone response elements or HREs. HREs are the sites of receptor binding to the regulatory regions of target genes, and these elements confer hormone inducibility to such genes. These elements generally act as enhancers in the sense that their influence on gene transcription is not strictly position- or orientation-dependent.

The DNA response element for the estrogen receptor is known as the estrogen response element or ERE. The consensus ERE, which is a perfect palindrome with the sequence GGTCA nnnTGACC, was initially discovered in the chicken (3) and Xenopus vitellogenin genes (4) and is thus often referred to as the vit-ERE as well as the consensus ERE. It is generally thought that the ER binds to the palindromic vit-ERE as a homodimer (5) . However, some recent data are more consistent with the estrogen receptor binding as a monomer (6) , and it is clearly established that other members of the steroid/thyroid superfamily can function as heterodimers (7) .

In addition to the vitellogenin ERE, a number of naturally occurring EREs have more recently been defined (8) , and most exhibit some sequence homology to the consensus element. However, there is considerable variability in these elements, and the vit-ERE is the only naturally occurring sequence so far identified that has two perfect palindromic half-sites. In contrast to the vit-ERE, few studies have directly investigated the nature of the estrogen receptor species that interacts with other EREs.

Although the majority of HREs identified to date are located in the 5`-flanking region of target genes within 1-2 kb or less of the promoter (9) , HREs have recently been found in the intron (10) and 3`-untranslated regions (11, 12) of hormone responsive genes. Thus, the location as well as the sequence of HREs for the steroid/thyroid receptor family may be more diverse than initially realized.

We and others (13, 14) have investigated the role of the AP-1 family of transcription factors as early response genes mediating estrogen-induced proliferation as this group of regulatory factors mediates proliferative responses to many other mitogens. These studies have shown that estrogen treatment produces rapid and dramatic in vivo increases in both c- fos (15, 16) and c- jun (17, 18, 19) expression in the rodent uterus, which result in large part from transcriptional activation (18, 19, 20, 21, 22, 23) . We and others have identified functional EREs in both the mouse (11, 24) and human (25) c- fos genes, but a HRE has not yet been identified in c- jun.

We now report the identification of an estrogen-dependent enhancer activity within the coding sequence of the rat c- jun. The sequence (GCAGA nnnTGACC) present in the c- jun exon competes with the consensus ERE for estrogen receptor binding in cell-free band shift assays and confers hormone inducibility to -galactosidase reporters in yeast and mammalian cell transcriptional assay systems. The identification of functional EREs in both major AP-1 partners coupled with their rapid induction by estrogen in vivo supports the hypothesis that AP-1 is a component of the estrogen-induced signal transduction cascade which regulates target cell proliferation.


EXPERIMENTAL PROCEDURES

Materials

DNA-manipulating enzymes were obtained from Boehringer Mannheim and Promega (Madison, WI). [-P]ATP was from Amersham Corp. Estradiol-17, mercaptoethanol, adenine sulfate, O-nitrophenyl-- D-galactopyranoside, uracil, and general reagents were purchased from Sigma. Dithiothreitol was from Boehringer Mannheim, whereas reagents for polyacrylamide gel electrophoresis were obtained from Bio-Rad. Casamino acids, yeast nitrogen base without amino acids, dextrose, and other media components were obtained from Difco. High performance liquid chromatography grade ammonium persulfate was obtained from Bio-Rad. Oxalyticase was obtained from Enzogenetics (Corvallis, OR). Reagents for polymerase chain reaction were from Perkin-Elmer.

Preparation of Soluble ER from Uterine Tissue

Uterine cytosol from 18-21-day-old Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, IN) was prepared as described previously (26) . Unless otherwise stated, estradiol-17- was added to a final concentration of 10 n M, and after a 1-h incubation in ice, cytosol was warmed to 25 °C for 45 min. To measure the ER content another aliquot of cytosol was labeled with 5 n M [H]estradiol in the presence or absence of 200-fold excess diethylstilbesterol for 4 h at 4 °C, and the reaction was terminated with dextran-coated charcoal as described previously (27) . ER concentrations generally ranged from 1 to 2 n M and were not altered by warming to 25 °C. Protein concentration was measured using the BCA kit from Pierce with bovine serum albumin as standard and generally ranged from 2 to 4 mg/ml. In some experiments, cytosol was used immediately for the band shift assay, and in others it was quickly frozen in liquid nitrogen for later use. There was no difference in results whether fresh or frozen cytosol was used for gel shift assays.

Band Shift Assays

Band shift assays utilized extracts from the rat uteri (prepared as described above) as the source of ER and double-stranded oligodeoxynucleotides. DNA was end-labeled with [-P]ATP, and T4-polynucleotide kinase was obtained from Stratagene (La Jolla, CA). Band shift assays utilized the basic approach described by us elsewhere (26) . Briefly, the binding reactions contained 10 m M Tris-HCl, pH 7.5, 50 m M NaCl, 1 m M dithiothreitol, 1 µg of poly(dI-dC) for every 5 µg of soluble protein and 0.05-0.20 ng of the vit-ERE probe (30,000-100,000 cpm) or 0.5-1.0 ng of the c- jun-ERE probe (approximately 200,000 cpm). The reactions generally contained 10 µg of the uterine protein extract as the source of receptor. In some experiments up to 40 µg of protein was used in order to help enhance binding of receptor to gene fragments derived from c- jun (not shown). The samples were incubated for 15 min at 4 °C before addition of the radioactive probe, and in most cases the reaction was continued for a further 15 min on ice. Following incubation the protein-DNA complexes were separated on 4% polyacrylamide gels in either 6.7 m M Tris-HCl, pH 7.5, 1 m M EDTA, 3.3 m M sodium acetate for samples containing binding reactions with radioactive c- jun-ERE or in Tris-glycine buffer obtained in the gel shift kit from Stratagene (La Jolla, CA) for experiments which involved ER interaction with radioactive vit-ERE only (competitive gel shift assays). Electrophoresis was conducted at a constant voltage of 160 V for 90 min, and the gels were then dried and autoradiographed.

The affinity-purified antibody ER-715 raised against residues 270-284 in the highly conserved hinge region of the ER (28) (kind gift of Dr. Jack Gorski) was used where indicated in the gel shift studies.

The double-stranded oligodeoxynucleotides used for the initial screening for ER binding in the competition band shift assays were the same as shown in Fig. 1except that they included a 4 base overhang at the 5`-end with GATC. The ERE from the Xenopus vitellogenin A2 gene (vit-ERE) was used as a standard ERE for comparative purposes (26) and is referred to as the ``consensus'' or vit-ERE throughout the text. Also the 3`- fos-ERE used for comparative purposes is shown in Ref. 26.


Figure 1: Structure of the rat c- jun gene and the location of ERE-like DNA sequences. The sequence data are from Ref. 46. The potential EREs were determined by sequence homology with the consensus ERE GGTCA nnnTGACC where n represents any base in the spacer arm between the two half-sites. Oligodeoxynucleotides used in competition gel shift assays to identify potential EREs in the c- jun gene contained additional GATC sequence in the 5`-end for cloning purposes. Sequences underlined show homology to the consensus or vit-ERE.



Plasmids and Plasmid Construction

Plasmid constructions and other cloning procedures were done using standard procedures (29) . The construction of YEPE10, a high copy number yeast, expression vectors for ER have been described previously (30) . These expression vectors were transformed into yeast strain BJ3505, and transformants were selected by tryptophan prototrophy.

Yeast reporter plasmids were constructed as follows. The yeast reporter plasmid YRPE2 was constructed from PLG670Z (31) by inserting a 75-base pair oligonucleotide (containing two estrogen-responsive elements) into the unique XhoI site upstream of the CYC1 (iso-1-cytochrome c) promoter. To construct the reporter plasmids, containing the c- jun-ERE-like elements and the mutant jun5 oligodeoxynucleotides, shown in Figs. 1 and 5, either single or double copies were inserted into the BglII site of the plasmid PC3 (32) . The copy number and orientation of the inserted oligodeoxynucleotides were determined by automated DNA sequencing.

To analyze the c- jun promoter in yeast for the presence of an ERE, the region between -1000 to -1 base pair from the start site of c- jun message was amplified from the plasmid containing -4500/+874 of the c- jun promoter (33) with primers containing a BglII recognition sequence. The conditions for polymerase chain reaction have been described previously (34) . Following digestion with the enzyme, this fragment was inserted into the BglII site of PC3, and the insert was sequenced for the determination of orientation. Another construct containing -440/-1 region of c- jun promoter was made by digesting the -1000/-1 polymerase chain reaction-amplified product with BamHI and ligating the resulting -440-base pair fragment into BglII-digested PC3 vector. These were also sequenced.

For transfection of mammalian cells, the Jun5ERE and the mutant Jun5EREs (Figs. 1 and 4) were inserted into pBLCAT2 (35) . Plasmids were sequenced and reporters containing two copies of the test sequence were selected for transfection studies.

Yeast Strains

The Saccharomyces cerevisiae BJ3505 ( MAT a, pep 4::HIS3, prb61-delta 1.6R, His3, Lys2-208, Trp1-delta101, Ura3-52, Ga12, (CUP 1)) was used throughout this study (36) . Yeast transformations were carried out using the lithium acetate polyethylene glycol transformation protocol (37) .

-Galactosidase Assays for Transcriptional Activity

Yeast cells transformed with the appropriate expression and reporter plasmids were grown overnight at 30 °C in selective medium (2% glucose, 0.5% casamino acids autoclaved to destroy endogenous tryptophan, 6.7 g/liter yeast nitrogen base, and 24 µg/liter adenine sulfate). This culture was used to inoculate fresh medium at a density of 100,000 cells/ml. The cells were incubated with 2 10 M estradiol. When cells reached an optical density of 1.0 at 600 nm, they were harvested and the level of -galactosidase produced was measured as described previously (38) .

Transfection of Mammalian Cells

H301 cells derived from estrogen-induced hamster kidney tumor were kindly provided by Dr. David Sirbasku (39) . Cells were grown in Dulbecco's modified Eagle's medium/F-12 supplemented with 10% fetal calf serum. For transfection studies H301 cells were plated on day 1 in complete medium. Twenty-four hours prior to transfection the medium was changed to Dulbecco's modified Eagle's medium/F-12 containing 5% dextran-coated charcoal-treated serum to remove endogenous steroids. Cells were transfected using the calcium phosphate method (11) with 10 µg of the reporter plasmid, 2 µg of the ER expression plasmid (40) , and 2 µg of the -galctosidase expression plasmid to monitor transfection efficiency. After 6 h cells were shocked with 10% glycerol and then treated with 20 n M estradiol for 24 h. Cells were then collected, lysed, and assayed for CAT activity. Results were corrected for the expression of -galactosidase activity and calculated for -fold induction of CAT activity in the presence of estradiol compared with control samples not treated with the hormone.


RESULTS

As noted in the Introduction, the protooncogene c- jun is rapidly induced by estrogens in the rat uterus in the presence of protein synthesis inhibitors (17, 41) , suggesting the gene is directly regulated at the transcriptional level by the interaction of the ER and an ERE. As an initial approach to search for potential EREs we examined the region of the rat c- jun gene which has been sequenced (-1388/+4144) for homology to the consensus element GGTCA nnnTGACC (see Fig. 1). This includes approximately 1.4 kb of 5`-flanking sequence, the single jun exon, and 2.2 kb of 3`-flanking sequence as shown in the Fig. 1. From this 5.7-kb region of DNA we selected fragments which had both: ( a) a 70% overall homology with the 10 nucleotides in the two arms of the consensus element and ( b) either one perfect half-site or 5 of the 6 bases which make important contacts during receptor-DNA binding interactions (42) . We developed these criteria since they would allow one to correctly select all the naturally occurring EREs reported (8) . This selection identified ten potential EREs: three in the 5`-flanking region, one in the 5`-untranslated region, two in the exon, and four in 3`-regions of the gene as shown in Fig. 1.

To test the gene fragments illustrated in Fig. 1for receptor binding, we synthesized double stranded 27-mer oligodeoxynucleotides containing the potential EREs (Fig. 1) in their naturally occurring contexts for in vitro binding studies. For these measurements we employed a sensitive gel shift competition assay as described previously (26, 43) . This assay measures the ability of excess unlabeled test oligodeoxynucleotides to competitively inhibit the binding of the ER to trace amounts of radioactively labeled consensus ERE sequence.

As shown in Fig. 2 A we tested the ability of a number of the potential jun EREs to compete for ER binding. Of those sequences tested in Fig. 2 A, only jun5 inhibits the binding of ER to the labeled consensus sequence. This sequence, GCAGA nnnTGACC, which is located in the single exon of the gene as illustrated in Fig. 1, contains one perfect half-site (TGACC), but 3 of 5 bases differ from the consensus sequence in the other half-site (GCAGA versus GGTCA). As also shown in Fig. 2A the unlabeled vit-ERE and the murine c- fos ERE (11) , which were used for positive controls, both compete for ER binding as expected.


Figure 2: Competition of ER binding to the vit-ERE by potential c- jun EREs. A, labeled vit-ERE (0.2 ng) was incubated with ER in the presence of either 50-fold molar excess of unlabeled vit-ERE, 1000-fold molar excess of the c- fos ERE, or 1000-fold molar excess of potential c- jun EREs shown in Fig. 1. B, gel shift assays similar to those in A showing a supershift of both the specific ER-ERE bands with ER antibody. Procedure for the gel shift assay is described under ``Experimental Procedures.''



In addition to the studies shown in Fig. 2 A, similar studies showed that jun2, jun4, and jun6 (see Fig. 1) do not compete for ER binding. Thus, of the 10 potential jun EREs illustrated in Fig. 1, only jun5 is able to bind the ER as judged by the competitive binding assay we utilized.

As shown in Fig. 2 A, two specific bands of ER binding to the labeled consensus sequence are observed. In some other experiments only one specific band was observed (data not shown). This is dependent upon the receptor preparation, irrespective of the presence of protease inhibitors in buffers (26) . Additionally a lighter nonspecific band ( NS) is observed with some receptor preparations. We have shown previously that the single specific band seen in some preparations is recognized by the antibody ER 715 to the ER (26) , thus establishing that the complex contains the hormone receptor. Fig. 2 B indicates that for those preparations exhibiting two bands, both are also recognized by antireceptor antibody. These bands do not appear to represent a monomer-dimer equilibrium, since when two bands are present their ratio remains constant over a 100-fold range of protein concentrations (data not shown). Others have occasionally observed multiple bands with gel shift assays using receptor preparations from the rat uterus (44) , and multiple species of ER may originate from single transcripts containing two translational start sites (45) . Most importantly for these studies both bands contain ER as indicated by the antibody supershifts, and both are competable with excess vit-ERE, fos-ERE, or the potential jun-ERE. The data in Fig. 2A indicate that the jun5 sequence (GCAGA nnnTGACC), which contains one half-site identical to the consensus sequence (GGTCA nnnTGACC), competes with the vit-ERE for receptor binding. We noted, however, that two other jun sequences (jun8 and jun9, Fig. 1) contain this identical half-site but fail to compete for ER binding, thus illustrating that not all sequences containing a perfect half-site compete for the binding of ER. This indicates that additional bases, either in the first half-site or immediately flanking the TGACC motif in jun5 have a significant influence on the ability of the jun5 ERE to bind the ER. The effect of bases immediately flanking this second half-site in jun5 is explored more fully in subsequent experiments (see Fig. 5).


Figure 5: Competition for binding of ER to the vit-ERE by native jun5 and with jun5 containing mutations. Mutations were in the first half-site (mt1jun5), the second half-site (mt2jun5), or upstream of the 5`-half site (mt3jun5). mt4jun5 and mt5jun5 contain mutations in the 5` and 3`, respectively, to the TGACC motif (see Fig. 4). Labeled vit-ERE (0.2 ng) was incubated with ER in the presence of either 1000-fold molar excesses of the unlabeled jun5 sequences with mutations shown in Fig. 4 or 50-fold molar excess of vit-ERE. Following incubation gel shift assays was performed as described under ``Experimental Procedures.''



Fig. 3 demonstrates that competition of jun5 for ER binding to the labeled vit-ERE is concentration-dependent. Increasing concentrations of both the homologous (vit-ERE) and the heterologous (jun5) oligodeoxynucleotides also show increasing competition for ER binding. In contrast, neither 500- nor 1000-fold excesses of the jun1 sequence, which is used here as a negative control, compete for ER binding. At these relatively high concentration of jun5 oligodeoxynucleotides, we have also noticed some competition for the nonspecific band (Figs. 2 and 3), although any possible significance of this observation is unclear at present. Studies such as those shown in Fig. 3 can also be used to estimate the relative binding affinity of the ER to different EREs (see ``Discussion'')


Figure 3: Concentration dependence for competitive jun5 binding to the ER. Labeled vit-ERE (0.2 ng) was incubated with ER in the presence of increasing molar excesses of unlabeled vit-ERE, the jun5 oligodeoxynucleotide or the jun1 oligodeoxynucleotide (see Fig. 1). Conditions for incubation and separation are described under ``Experimental Procedures.''



Based on these results with a competition assay, we attempted to directly observe the binding of the radioactively labeled jun5 oligodeoxynucleotide with the ER. However, we could not observe a direct binding interaction of this element with the ER despite using several experimental modifications of our basic binding assay (26) . These modifications included preloading of the receptor with estradiol-17, increasing the preincubation time of the receptor with the jun5 oligodeoxynucleotide from 15 min to 24 h before electrophoresis, or increasing the protein concentration of the receptor preparations from 10 to 40 µg/sample during the incubation (data not shown). These results indicate that the affinity of the ER for the jun5 sequence is insufficient to maintain complex formation through the electrophoresis step in such direct binding measurements.

To further explore the role of specific nucleotides in the ER-jun5 binding interaction, we synthesized a series of oligodeoxynucleotides containing various mutations in the two-half sites and various flanking regions of the element. As shown in Fig. 4, these sequences contain mutations in the GCAGA first half-site (mt1jun5), the TGACC second half-site (mt2jun5), mutations 5` of the GCAGA half-site (mt3jun5), mutations in the spacer region between the two half sites (mt4jun5), and mutations 3` of the TGACC half-site (mt5jun5). These sequences were used in the competition studies illustrated in Fig. 5.


Figure 4: Oligodeoxynucleotides with mutations in the putative jun5ERE sequence were prepared for use in the competitive binding studies in Fig. 5.



The overall pattern of mutational effects on ER-jun5 binding (Fig. 5) was somewhat surprising to us. As expected, mutations in the CC dinucleotide of the second half-site (TGACC to TGAtt) led to a loss of receptor binding as evidenced by the lack of competition with mt2jun5. However, mutations in the first half-site of the element (GCAGA to ttAGA) had no observable effect as the mt1jun5 sequence was able to compete as effectively as the jun5 parent element. Similarly, mutations 5` to the GCAGA half-site (mt3jun5) had no apparent effect on receptor binding.

These results indicate that the first half site (GCAGA) of the jun5 element has little, if any, effect on receptor binding, whereas mutations in the TGACC sequence destroy binding. This observation alone might imply that only the second half site is involved in ER binding, but this is inconsistent with the observation that this TGACC motif when present in other regions of the gene (see jun8 and jun9 sequences in Fig. 1) does not appear to bind the receptor (Fig. 2). This suggested that nucleotides immediately adjacent to the TGACC motif contribute to the specificity of ER binding to jun5.

Further results illustrated in Fig. 5indicate that the replacement of two nucleotides immediately 5` (mt4jun5) or immediately 3` (mt5jun5) to the TGACC half-site destroy the ability of oligodeoxynucleotides to compete for ER binding. This suggests that the TGACC motif and its immediate flanking bases are sufficient to confer the receptor binding observed in our competition gel shift assays.

Given these findings in cell-free receptor binding ER studies, it was next of interest to test the ability of jun sequences to confer hormone-inducible functional responses in intact cell systems. For this purpose we initially utilized a yeast-based transcriptional assay, because it displays very low levels of background activity in the absence of estrogens. Fig. 6 A represents results from a series of experiments illustrating that the jun5 ERE is capable of directing hormone-dependent transcription when present upstream of reporter constructs in either the positive or negative orientation. As for many other enhancers, the effect of the jun5 ERE on hormone-dependent induction of the -galactosidase reporter activity is synergistic if multiple copies are present. Measurements of inducibility conferred by the vit-ERE are also included as a positive control, and as expected this element is a powerful inducer of reporter activity. In addition we utilized a plasmid containing the sequence GGTCT nnnAGACC as a negative control (labeled GGTCT in Fig. 6A). We previously showed that this sequence does not confer hormone inducibility in either mammalian cells (11) or in yeast (43) despite its high degree of homology to the vit-ERE (GGTCA nnnTGACC).


Figure 6: A, Jun5 confers estrogen-dependent transcriptional activation. Either single or double copies of the jun5 oligodeoxynucleotide were ligated to the yeast-based vector in positive and negative orientations and used to transform yeast. Vectors containing two copies of vit-ERE or an inactive ERE-like sequence from c- fos promoter (GGTCT nnnAGACC; see Ref. 43) were included as positive and negative controls respectively. In all cases, yeast were cotransfected with an ER expression plasmid. Yeast were then incubated in the absence ( -H) or presence ( +H) of estradiol and -galactosidase activity measured as described under ``Experimental Procedures.'' B, mutations in either half-site of the jun5ERE destroy estrogen-dependent transcriptional activity. Vectors containing two copies of either the native jun5ERE mutations in either the first (mt1jun5) or the second half-site (mt2jun5) of the jun5 sequence were cotransfected into yeast with an ER expression plasmid and tested for estrogen-dependent activity as described in A.



In a companion series of measurements, we next explored the effects of mutations in either the first or second half site of the jun5 sequence on transcriptional activity (Fig. 6 B). As expected, the mutation in the TGACC motif (mt2jun5) which destroys receptor binding activity (Fig. 5) also abolishes hormone inducibility (Fig. 6). Surprisingly, however, the mutation in the first half-site (mt1jun5) which has no effect on receptor binding (Fig. 5), causes an almost complete loss of hormone inducibility (Fig. 6). This unexpected finding indicates that the first half site of the jun5 sequence is crucial for estrogen-dependent transcriptional activation, but is not required for receptor binding per se. This strongly suggests that the interaction of a factor other than the ER at the GCAGA region of the jun5 element is necessary for the hormonal induction of transcriptional activity.

Given these findings in yeast, it was important to determine if the jun5 sequence was also active as an ERE in an estrogen-responsive mammalian cell line. We thus transfected reporters containing this element into the estrogen-responsive hamster cell line H301 as described under ``Experimental Procedures.'' In three separate determinations, estrogen caused a highly significant ( p < 0.001) induction of CAT activity (2.72-fold ± 0.33 S.E.) in constructs containing the jun5ERE. Estrogen did not cause a significant induction of the CAT activity in the control vector (pBLCAT) or the constructs containing mt1jun5 or mt2Jun5. In fact, the hormone appeared to slightly decrease CAT expression from both mutant constructs. Although these results differ quantatively from those seen in the yeast system, they establish that the jun5ERE confers hormonal inducibility in estrogen-responsive mammalian cells.


DISCUSSION

c- jun is rapidly and transiently induced by estradiol in the uterus of ovariectomized rats (17, 19, 41) . Nuclear run-on assays (23) and the insensitivity of the induction to protein synthesis inhibitors (17, 41) indicate that jun induction is a primary hormonal response mediated by the interaction of the ER with cis-regulatory elements of the gene. The major goal of this study was thus to determine if c- jun contains a functional ERE(s).

To identify potential EREs in rat c- jun, we initially scanned the entire known nucleotide sequence of the gene (46) for regions homologous to the consensus vit-ERE, GGTCA nnnTGACC. Based upon this strategy, 10 candidate sequences were identified and screened for ER binding with a competitive gel shift assay that tests potential EREs for their ability to inhibit the binding of the ER to the consensus. This is a useful screening approach, because it can identify elements with a wide range of affinities for the receptor (26, 43) . Of these candidate EREs only the sequence GCAGA nnnTGACC (jun5) present in the single exon of the gene was active in this test system (Fig. 2). This sequence contains a perfect second half-site, but has three mismatches from the consensus ERE in the first half site. This is similar to the c- fos ERE previously identified which also contains one perfect consensus half-site with three mismatches in the second (11) .

Using the competitive binding approach (Fig. 3) we estimate that the relative affinity of the ER for the ERE in jun5 is approximately 3-7-fold less than its affinity for the murine fos ERE (26) and 100-200 fold less than that measured for the vit-ERE. Although it may be entirely fortuitous, it is interesting to note in this regard that in vivo dose-response studies have shown that the EDvalue for estradiol induction of c- jun mRNA in the uterus is about 3-4-fold higher than the corresponding value for c- fos induction (17) .

Estimates of the Kof the vit-ERE for the estrogen receptor are approximately 0.1 n M (6) which in turn suggests a very approximate Kof 10-20 n M for the c- jun sequence present in jun5 and 1-10 n M for the c- fos ERE. Given that the estrogen receptor content of uterine cells has been estimated at 30 n M (47) , these affinities are consistent with a potential physiological role for these c- fos and c- jun EREs in estrogen-induced proliferation. Furthermore, it is important to remember that these rough estimates of affinity are based solely on cell-free binding studies using small pieces of naked DNA. In vivo, the tertiary structure of DNA, neighboring regions of chromatin/DNA, additional transcription factors, other proteins, etc. may have significant influences on ER-ERE interactions. This seems especially likely for the putative jun ERE identified in this work, since differences between the residues required for binding and transcriptional activation suggest the involvement of another factor in receptor binding to this element (discussed more fully below).

To determine if the ER binding to jun5 observed in cell-free studies has any functional significance, we initially used a well established yeast based transcriptional assay system that has successfully identified EREs in other estrogen-regulated genes (43 and references therein). Plasmids containing both single and double copies of the jun5 element increase hormone-dependent reporter activity (Fig. 6). As frequently seen for other regulatory sequences, multiple copies of the jun5 element yield synergistic increases in hormone inducibility. This may suggest that the jun5 sequence interacts with other regulatory elements/factors to control the overall expression of c- jun. The results in Fig. 6also indicate that the enhancement of transcriptional activity is independent of the orientation of the jun5ERE, fulfilling the criteria for an element to be considered a traditional enhancer. Further transfection studies in H301 cells, an estrogen-responsive hamster tumor cell line (39) , established that the rat c- jun ERE also confers hormone inducibility in mammalian cells as well as yeast. This finding is in agreement with a recent report showing that a number of isolated EREs are active in both the yeast and mammalian cells (48) .

Both the receptor binding and transcriptional activation studies indicated that the jun5 sequence located within the single exon of the gene can function as an ERE. However, most EREs identified to date have been found upstream of target gene promoters (9) . We thus investigated whether the 5`-flanking region of c- jun contains additional sequences with ERE activity which were not selected in our original screen that was based on homology to the consensus element. For this purpose we constructed a yeast plasmid containing a 1-kb fragment (-1/-1000) of the 5`-flanking region of the c- jun gene as described under ``Experimental Procedures.'' This fragment was totally devoid of estrogen-inducible activity in experiments analogous to those performed in Fig. 6. These findings do not unequivocally rule out the possibility of other EREs in the distal 5`-flanking region of c- jun.

To determine if the element we identified in the rat is present in other species, we examined the mouse and the human c- jun genes for similar sequences. The second half site of the rat element and the two immediate flanking bases on either side (CATGACCTT) are completely conserved in the mouse gene (49) and differ by only one purine substitution in a flanking nucleotide (CATGACCcT) in the human gene (50) . The conservation of this sequence and the estrogenic regulation of jun expression in target cells from all three species further supports a role for this element in the hormonal control of this early response gene.

Experiments aimed at investigating the precise structure-function relationships for receptor binding and transcriptional activation of the GCAGA nnnTGACC sequence present in jun5 provided unexpected results. While mutations in the TGACC motif abolished receptor binding, mutations in the GCAGA half-site as well as those 5` to that half-site did not seem to affect ER binding in vitro (Fig. 5). This strongly suggests that the ER does not bind to the jun5 sequence as a homodimer, which is the form generally considered to bind to the palindromic vit-ERE (5) .

Nucleotides immediately flanking the TGACC pentanucleotide are important for ER binding (see mt4jun5 and mt5jun5 in Fig. 4), as recently reported for other hormone response elements. For example, not all gene fragments containing the consensus ERE bind the receptor with high affinity (51) , and neighboring bases are involved in the differential ERE-receptor interactions when the receptor is liganded with estrogen versus anti-estrogen (8) . Along with our findings, these results suggest that bases immediately adjacent to receptor binding sequences have a greater impact on the binding interaction than previously recognized.

In parallel experiments we also examined the effects of mutations in the jun5ERE on transcriptional activation. In contrast to the receptor binding studies, mutations in either half site of the GCAGA nnnTGACC sequence destroy the ability of the element to confer estrogen responsiveness to reporter constructs. This was a surprising result which emphasizes that different sets of nucleotide interactions mediate ER binding under cell-free conditions and transcriptional activation in transfection studies.

The combined results of these structure-activity relationships for the receptor binding and transcriptional activation could be interpreted in several ways. One possibility is that the direct binding of the ER to the jun5 sequence observed in our cell-free measurements does not play a role in c- jun induction in intact cells, but the ER is involved indirectly via protein-protein interactions analogous to those controlling ovalbumin expression (52) via a single consensus half-site (GGTCA). This possibility would seem more likely if both half-sites of the jun5 sequence were needed for binding in cell-free studies, and only one site was necessary for hormonal inducibility. Our results, however, are just the opposite. Furthermore, in the ovalbumin system there is no evidence for binding of the ER to a half-site similar to that seen in jun5, and hormonal inducibility requires only one ovalbumin half-site rather than both half sites as with jun5.

We believe the most likely interpretation of our data is that the ER interacts with an unidentified protein factor to regulate estrogen induction of c- jun expression. In this scenario, the ER binding involves primarily the TGACC half-site with additional stabilization from its flanking dinucleotides, and another protein factor would interact primarily with the GCAGA half-site. This interaction could occur either as the binding of a preformed ER heterodimer to the GCAGA nnnTGACC sequence, or the ER and the other postulated protein factor could bind to the DNA independently and then interact to stimulate transcription.

Such a heterodimerization model is also attractive in light of recent studies showing that induction of c- jun in the uterus exhibits a cell-specific pattern of expression (53, 54) . All major uterine cell types contain estrogen receptors (55) , and estrogens elicit responses in all these cell layers of the immature rat uterus (56) . However, estrogen treatment in vivo leads to a decrease in c- jun expression in uterine luminal epithelial cells, an increase in c- jun expression in glandular epithelial cells and some myometrial cells, and little apparent change in most uterine stromal cells (54) . This combined pattern of estrogen receptor distribution and hormonal induction of c- jun expression in the uterus suggests that factors besides the ER are involved in the estrogenic regulation of the protooncogene in this hormone target organ.

While regulation of c- jun expression by a heterodimeric complex of the ER and another protein is an attractive possibility, we have no information about the possible identity of a non-receptor partner. We have tested several known factors, including AP-1 components (57) , Nur77, and Nur1 proteins (58) , for binding to jun5, since their consensus DNA binding sequences (TGACTCA and AAAAGGTCA, respectively) show some similarity to sequences in this region of jun, but these results were negative. Although attractive, the suggestion that a heterodimeric ER controls jun expression is thus hypothetical at present. Furthermore, our results in no way exclude the possibility that other factors or elements play a role in the physiological regulation of c- jun expression by estrogens.

In summary, we have identified the sequence GCAGA nnnTGACC within the exon of rat c- jun that binds the ER and confers hormonal inducibility to reporter genes, properties expected of a physiological hormone response element. To our knowledge this is the first example of an ERE within the coding sequence of an estrogen-inducible gene, although there is clear evidence for such regulatory elements in the coding region of other genes (59) . Structure-function studies are most consistent with a model in which this element regulates transcription by interacting with a heterodimer containing the ER and another protein component. The presence of a functional ERE in c- jun further supports a role for AP-1 as an early component of the estrogen-induced proliferative response in target tissues such as the uterus.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant HD-08615. 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) X17215.

§
To whom correspondence should be addressed: Dept. of Pharmacology, University of Texas Medical School, P. O. Box 20708, Houston, TX 77225. Tel.: 713-792-5967; Fax: 713-792-5911.

The abbreviations used are: ER, estrogen receptor; HRE, hormone response element; ERE, estrogen response element; kb, kilobase(s); CAT, chloramphenicol acetyltransferase.


ACKNOWLEDGEMENTS

We are grateful to Dr. Jack Gorski and the National Institutes of Health for the provision of the estrogen receptor antibody ER-715 used in this study. We thank Drs. Evelyn Murphy and Orla Connealy from Baylor College of Medicine for provision of Nur1 and Nur77 proteins for gel shift assays. We are grateful to Dr. Ming Tsai for the provision of estrogen receptor expression plasmid.


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