From the
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.
The estrogen receptor (ER)
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
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.
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.
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.
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).
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.
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
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.
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 ED
Estimates of the K
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.
The nucleotide sequence(s) reported in this paper has been
submitted to the GenBank/EMBL Data Bank with accession number(s)
X17215.
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
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.
-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.
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.
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 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) .
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 -Galactosidase Assays for Transcriptional
Activity
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.
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.
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.
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.
-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.
value 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) .
of the vit-ERE for
the estrogen receptor are approximately 0.1 n
M (6) which in turn suggests a very approximate
K
of 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).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.