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(Received for publication, July 5, 1995; and in revised form, November 20, 1995) From the
We have shown previously that estrogen-stimulated transcription
from the human lactoferrin gene in RL95-2 endometrium carcinoma cells
is mediated through an imperfect estrogen response element (ERE) at the
5`-flanking region of the gene. Upstream from the ERE, a DNA sequence
(-418 to -378, FP1) was selectively protected from DNase I
digestion by nuclear extracts from endometrial and mammary gland cell
lines. In this report, using the electrophoresis mobility shift assay,
site-directed mutagenesis, and DNA methylation interference analyses,
we show that three different nuclear proteins bind to the FP1 region
(C1, C2, and C3 sites). The nuclear receptor, COUP-TF, binds to the C2
site. Mutations in the C1 binding region abolish C1 complex formation
and reduce estrogen-dependent transcription from the lactoferrin ERE.
When the imperfect ERE of the lactoferrin gene is converted to a
perfect palindromic structure, the enhancing effect of the C1 binding
element for estrogen responsiveness was abolished. We isolated a
complementary DNA (cDNA) clone from an RL95-2 expression library that
encodes the C1 site-binding protein. The encoded polypeptide maintains
99% amino acid identity with the previously described orphan nuclear
receptor hERR1. A 2.2-kilobase mRNA was detected in RL95-2 cells by the
newly isolated cDNA but not by the first 180 base pair of the published
hERR1 sequence. By Western analysis, a major 42-kDa protein is detected
in the RL95-2 nuclear extract with antibody generated against GST-hERR1
fusion protein. Finally, we show that the hERR1 interacts with the
human estrogen receptor through protein-protein contacts. Eukaryotic gene promoters consist of multiple upstream
regulatory elements that positively or negatively modulate
transcriptional activity (for review, see Yamamoto(1985)). For the
steroid hormone-responsive gene, the regulation is mediated through
steroid receptor binding to its respective hormone response element
(HRE) ( We have been studying lactoferrin, an estrogen-inducible gene
product present in milk, tears, and saliva (Teng et al., 1989
and references therein). Lactoferrin has multiple functions that
include modulating the immune response, promoting cell growth, and
killing bacteria (Arnold et al., 1976; Broxmeyer et
al., 1987; Esaguy et al., 1991; Legrand et al.,
1992; Nichols et al., 1987; Sawatzki and Rich, 1989). Although
the lactoferrin gene is expressed in many tissues, its expression in
the mouse uterus is very sensitive to estrogen (Pentecost and Teng,
1987; Teng et al., 1989); estrogen injection into a 21-day-old
mouse induces lactoferrin messenger RNA several hundred-fold (Pentecost
and Teng, 1987). Accordingly, uterine lactoferrin protein and messenger
RNA fluctuate with plasma estrogen levels during the estrus cycle
(Newbold et al., 1992; Walmer et al., 1992).
Expression of lactoferrin gene in human endometrium, however, is not
nearly as robust as that in the mouse uterus (Teng et al.,
1992; Walmer et al., 1995). Comparisons of the
promoter/enhancer region from human and mouse lactoferrin genes
revealed a similar composite estrogen response element (Teng et
al., 1992; Teng, 1994). The mouse lactoferrin ERE overlaps a
functional COUP-TF binding element (Wang et al., 1989, 1991),
generating a direct competition between these two transcription factors
for binding to their overlapping regions of the element (COUP/ERE
element) (Liu and Teng, 1992; Liu et al., 1993). We
demonstrated that overexpression of COUP-TF in transfected uterine
endometrial cells repressed estrogen stimulation (Liu et al.,
1993). The human and mouse lactoferrin COUP/ERE elements are located at
similar positions upstream from the start site and are well matched (18
of 22 nucleotides identical) (Liu and Teng, 1991; Teng et al.,
1992; Teng, 1994). In contrast, COUP-TF binds DNA elements different
from COUP/ERE in the human lactoferrin promoter (Teng et al.,
1992; Yang and Teng, 1994). Recently, we found a DNA sequence
(-414 to -378, FP1) upstream from the COUP/ERE that was
selectively protected from DNase I digestion by nuclear extracts of
endometrial (RL95-2) and mammary gland (HB100) cell lines (Yang and
Teng, 1994). We defined an extended steroid receptor half-site,
TCAAGGTCATC, within the FP1 that matches the consensus binding elements
of SF-1/ELP (Ikeda et al., 1993; Tsukiyama and Niwa, 1992).
These tissue-specific transcription factors belong to a nuclear
receptor subfamily that bind as monomers (Wilson et al., 1993;
Ikeda et al., 1993; Tsukiyama and Niwa, 1992). Since different
transcription factors may bind to identical response elements in
various cell types, we sought the nuclear factors in RL95-2 cells that
bind to this DNA element. In this study, we mapped the nuclear protein
binding elements in the FP1 region and demonstrated that the
TCAAGGTCATC element enhances estrogen responsiveness of the human
lactoferrin gene. Subsequently, an RL95-2 expression library was used
to isolate cDNA that encodes another binding protein for the extended
steroid receptor half-site. The cDNA clone was sequenced and identified
as hERR1 (Giguere et al., 1988). Furthermore, we showed that
the hERR1 interacted with estrogen receptor through protein-protein
contact, suggesting that ERR1 may participate in estrogen stimulation
of human lactoferrin gene.
Figure 1:
Specific
interaction between RL95-2 nuclear protein and FP1 region. A,
diagrammatic presentation of the location of the DNase I footprint
region, probes, and the competitors of the human lactoferrin gene used
in this study, position of the RL95-2 nuclear protein-DNA complexes
(C1, C2, and C3) and the mutated Gs (m1, m2, and m3) in FP1 are
indicated. B, FP1 and nuclear protein of the RL95-2 cells
formed three specific protein-DNA complexes in EMSA (marked at the left side as C1, C2, and C3). Fifty-fold molar excess of cold
oligonucleotide -418/-378 (FP1) was used in the competition
(lane marked as +).
Figure 3:
Mutations at the DNA contacts in FP1
region abolished protein-DNA interaction. Three µg of protein were
interacted with various labeled probes, and the reaction products were
analyzed in 3.5% nondenaturing polyacrylamide gel in EMSA. A,
interaction between the RL95-2 nuclear proteins and the wild type or
the mutated FP1 (-418/-378) probe. B, location of
the mutated Gs in various oligonucleotides. C, specific
COUP-TF antibody interacted with the C2 complex. COUP-TF antibody
supershifted band are labeled as SB. Protein-DNA complexes,
C1, C2, and C3 are indicated. A nonspecific band (NS)
appearing with the m2 probe is marked. Free, free
probe.
The RL95-2 expression cDNA library was screened with
labeled FP1 oligonucleotides (1.47 kilobase pairs of the pSLFP-32
insert) as described (Vinson et al., 1988). The
protein-expressing clone interacting with the DNA probe was isolated
and designated as FP1.4. Double-stranded nucleotide sequencing of the
FP1.4 was performed in our laboratory and by Lark Sequencing Co.
(Houston, TX). Four additional positive clones, two from the
RL95-2 expression cDNA library (FP1.4-2 and FP1.4-3) and two from
the human hippocampus expression cDNA library (HP1 and HP2)
(Stratagene, La Jolla, CA), were subjected to additional DNA sequencing
at regions (from nucleotides 740 to 760 and 1190 to 1250) diverging
from the published hERR1 (Giguere et al., 1988).
The Western blots were probed by antiserum either
to GST-hERR1 or to ER (H222, Abbott, Chicago, IL) with an ECL kit
(Amersham Corp.) according to the manufacturer's specification.
The far-Western technique was performed as described (Kaelin et
al., 1992) with
Figure 6:
Identification of hERR1. A,
nucleotide sequence and deduced amino acids of hERR1. The nucleotide
sequence and the longest open reading frame of hERR1 were presented.
The different nucleotides from published hERR1 were indicated on top of the sequence and the amino acids on the bottom. The two zinc-fingers were boxed. B,
northern blot analysis of hERR1 mRNA in RL95-2 cells and mouse tissues. A, 2 µg of poly(A) RNA from RL95 cells and from mouse
kidney tissue were analyzed. The 5`-specific probes were described
under ``Materials and Methods.'' Lanes 1 and 2 were probed with 180R of hERR1 (Giguere et al., 1988). Lanes 3 and 4 were probed with 185B of hERR1 (Fig. 1). The same blots were reprobed with
Figure 2:
Map of the RL95 nuclear protein contacts
in FP1 by methylation interference analyses. The RL95-2 nuclear
protein-DNA complexes were resolved in 3.5% nondenaturing
polyacrylamide gel. The individual bands were excised from the gel, and
the methylation interference analyses were performed. A,
methylation interference analysis. Location of the DNA contacts from
each complex is indicated at either side of the gel. The solid
symbol represents strong contacts, and the open symbol represents weak contacts. B, position of the DNA
contacts.
To
further examine the binding sites for the proteins that interacted with
FP1, we performed EMSA using mutated oligonucleotides (Fig. 3).
In agreement with the methylation interference findings, mutations (G
to C) at the C1 and C3 contacts (m1 and m3, respectively) abolished the
protein binding at these regions (Fig. 3A, lanes 2 and 12, respectively), whereas a mutation at the noncontact Gs
(m2) did not interfere with protein-DNA interaction (Fig. 3A, lane 7). The competition experiments
demonstrated that the oligonucleotide -418/-378 containing
the entire FP1 region (Fig. 1) competed for binding with all
three complexes (Fig. 3A, lanes 4, 9, and 14).
Oligonucleotide -375/-340, however, containing the COUP-TF
binding element and the imperfect ERE competed for C2 (lanes 5,
10, and 15). The oligonucleotide -418/-394,
which covered the 5` half of the FP1 region, competed with C3 (lane
6 and 11). As expected, mutation at the C1 contact sites
(m1) weakened the C2 binding (compare lanes 1 and 2),
since this was also the C2 binding region. Unexpectedly, under this
condition, oligonucleotide -418/-394 could compete with C2,
but not if the C1 contacts were intact (compare the intensity of C2 in lane 6 to lanes 11 and 16). Similarly, C3
binding was influenced by mutation at C1 contact sites (compare the
intensity of C3 in lanes 2 and 5). C2 binding was not
affected by mutations at other locations such as m2 and m3 (compare the
intensity of C2 in lanes 7, 11, 12, and 16 with lane 1). By using specific COUP-TF antibody in the EMSA, the
C2 complex was supershifted (Fig. 3C, lane 2 and 4). Although the C2 binding was substantially reduced with m1
oligonucleotide as the probe, COUP-TF antibody interacted with the C2
complex. This observation confirmed the competition experiments (Fig. 3A) that COUP-TF is present in the C2 complex.
Figure 4:
The
extended steroid receptor half-site enhances ER-mediated estrogen
responses. Wild type and mutated pHL-414CAT reporter constructs were
cotransfected with ER expression plasmid (HEO) to the RL95-2 cells.
Cells were treated with or without diethylstilbestrol for 24 h before
harvest. A, effect of mutations at m1, m2, and m3. B,
effect of mutations at m1 and m6 (destroying the imperfect ERE as
marked by X in the dotted box). C, effect of
mutations at m1 and m7 (converting the imperfect ERE to palindromic ERE
as marked by the striped box). Different lots of RL95-2 cells
from ATCC were used in experiments A and experiments B and C. The CAT activity was determined by thin-layer
chromatography and PhosphorImager system. Each set of the experiments
has been repeated five times in duplicate. The results were normalized
with protein concentration and presented as means ± S.D. Fold of
stimulation is indicated on top of the bar.
When the
double Gs in the conserved steroid receptor half-site (C1 binding
region) were changed to Cs in m1-CAT reporter constructs (Fig. 3B), the estrogen stimulation was reduced
significantly (Fig. 4A, compare wt and m1). Although the basal activity of m1 was slightly lower than
wild type, the estrogen-stimulated CAT activity was affected more by
mutation at this region. By using different lots of RL95-2 cells we
found variations in both basal and estrogen-stimulated CAT activities
(compare wt in Fig. 4, A-C). Despite
this variations, mutation at C1 binding region consistently showed
2-fold reduction in estrogen responsiveness (compare wt and m1 in Fig. 4, A and B). It was
interesting to find that destruction of ER binding to the ERE (m6) did
not attenuate estrogen-stimulated activity completely unless C1 binding
was also destroyed at the same time (m1/m6). These results suggest that
both C1 and the imperfect ERE in the human lactoferrin gene are
required for maximum strength of estrogen induction. The question
arises as to whether the C1 dependence could be abolished by a
palindromic ERE, which is a stronger enhancer than the imperfect ERE in
the lactoferrin gene. To test this possibility, we converted the
imperfect ERE to a palindromic ERE (m7) in the pHL414CAT reporter
construct containing an intact or a mutated C1 (m1). These reporter
constructs were transfected into RL95-2 cells, and the estrogen
responses were examined (Fig. 4C). When the imperfect
ERE was converted into a perfect ERE, the strength of estrogen action
was doubled (Fig. 4C, compare wt and m7). Destroying the C1 has no effect on estrogen-stimulated
activity (compare m7 to m1/m7). Therefore, only the
weak imperfect ERE needs extra help from C1 to confer ER-mediated
activity, whereas a strong ERE can function independently.
Figure 5:
C1 complex is responsible for the enhanced
ER-mediated activity. A, identify the critical nucleotides
involved in C1 binding. Various mutated oligonucleotides are used as
competitors in the band shift assay. The mutations are indicated. The shaded nucleotides belong to the linker. B, effect of
mutant d on estrogen responsiveness.
Comparison of the FP1.4 to the hERR1 sequence indicated that
there were seven deletions and one mutation in the coding region and
two deletions and one addition at the 3`-noncoding region of the clone.
Deletions occurring in the coding region caused frameshift mutations
and generated three areas of amino acid discrepancy from the published
hERR1. The differences in amino acids were marked at the bottom of the
sequence (Fig. 6A).
From the Western blot analysis, antibody produced
against the hERR1 fusion protein detected a 42-kDa protein from human
uterine and mammary gland cell lines (Fig. 6C). A minor
protein band at the 53-kDa region was also detected in these cell
lines. In HeLa cells, there were equal amount of 53- and 42-kDa
protein. The predominant protein expressed by mammary gland cells from
both human (HBL100) and mouse (comma-D) was the 42-kDa protein.
Figure 7:
Identification of the contact sites of
GST-hERR1 fusion protein at the FP1 of the human lactoferrin gene. A, specific binding of GST-hERR1 to FP1 oligonucleotides in
EMSA. One µg of GST-hERR1 fusion protein was interacted with
preimmune serum (PI) or hERR1 antibody (ERR1) before
incubation with the labeled FP1 (-418/-378) in 10 µl of
reaction mixture. B, methylation interference analysis. Five
ng of labeled and partially methylated DNA fragment (FP1) was incubated
with 10 µg of GST-hERR1 fusion protein in 60 µl of reaction
mixture. The locations of GST-hERR1 contacts on both DNA strands are
indicated. The solid symbols represent strong contacts, and
the open symbols represent weak contacts. The sequence is
presented at the bottom.
Figure 8:
Immunodepletion of the C1 complex by
antibody produced against the GST-hERR1 fusion protein. Three µg of
nuclear protein from RL95-2 cells was incubated with labeled FP1 in an
EMSA. Preimmune serum (PI), affinity-purified hERR1 antibody (ERR1), Vit-A2 ERE, oligonucleotide of vitellogenin
A2 estrogen response element and free probe F are
indicated.
Figure 9:
Direct interaction of hERR1 and ER.
Bacteria expressed various fusion proteins (2 µg, lanes 3, 4,
6-8 and 11, 12, 14-16; 5 µg, lanes 5 and 13) were separated on a 10% SDS-PAGE, Western
blotted, and hybridized with
We mapped the C1, C2, and C3 proteins binding sites (Fig. 1Fig. 2Fig. 3) through a series of
experiments to characterize the proteins that bind FP1 region of the
human lactoferrin gene. We confirmed our previous finding that COUP-TF
binds C2 (Yang and Teng, 1994). The C3 protein and its DNA binding
element were investigated but not characterized in this study. The
EMSA, transient transfection, and site-directed mutagenesis studies
showed a correlation between C1 binding to the DNA element,
TCAAGGTCATC, at the 3` end of the FP1 region and up-regulating the
estrogen response of the human lactoferrin gene. The functional studies
were conducted in transiently transfected human endometrial carcinoma
cells. An inherent problem of transient transfection experiments is the
changing basal promoter strength in mutant constructs (Fig. 4).
An aberrant initiation of transcription or lost binding of the positive
or negative transcription factors that are part of the basal promoter
machinary might contribute to the variable basal promoter activities.
In addition, transfection experiments carried out with cells in various
passages and different lot numbers could have inconsistent basal
activities. Obviously, these changes will also affect
estrogen-stimulated activities (Fig. 4A). Despite these
variables, the estrogen responsiveness of the human lactoferrin
promoter is unchanged in reporter constructs having mutations outside
the C1 binding site (compare fold of stimulation in wt to m2 and m3). Mutations within the C1 binding site have
significant effect on estrogen responsiveness, regardless the basal
promoter strength (compare fold of stimulation in wt to m1 in Fig. 4, A and B). Mutant d exclusively
prevents formation of the C1 complex showed reduced estrogen response
in transient transfection experiments (Fig. 5). These results
provided further support for an important role of the C1 protein.
Collectively, information from the EMSA and transfection experiments
strongly suggests that C1 binding is important in maximizing estrogen
stimulation. We isolated the cDNA that encodes C1-binding protein,
and by sequencing, we verified that it is hERR1 (Fig. 6A). Several internal deletions in the hERR1
coding region predicted an amino acid deletion at nucleotide 746 and a
frameshift at 1208-1236, which caused 10 mismatched amino acids
in the potential ligand binding region. These differences may be
significant in terms of ligand binding. The polypeptide encoded by
hERR1 was tested for steroid binding capability, but none were found
(Giguere et al., 1988). Changes of amino acid sequence in the
potential ligand binding domain of the hERR1 could render ligand
binding. The apparent differences between the published and our hERR1
sequences lies at the 5` end. 2.2-Kilobase hERR1 mRNA in RL95-2 cells
and mouse kidney, detected by the 5` probe of our sequence (nucleotides
1-185, Fig. 6A), but not by the 5` probe of
published sequence (nucleotides 1-180; Giguere et al.,
1988), suggests a truncated hERR1 mRNA in these cell and tissue.
Examining the published hERR1 cDNA sequence, nucleotides 1-178
originated from By using hERR1 as a probe, we isolated several cDNA
clones from mouse brain and kidney cDNA libraries. ( Examining the distance between the hERR1 and ER binding sites
(center to center) in the lactoferrin promoter, we found that there are
three DNA helical turns between them (Teng et al., 1992). It
is possible that hERR1 and ER both bind to their DNA element on the
same side of the helix and interact with each other through a direct
protein-protein contact. Indeed, by far-Western analysis, we were able
to demonstrate protein-protein contact between hERR1 and ER (Fig. 9). Interaction between ER and other nuclear proteins has
been found. Several lines of evidence suggest that AP-1-binding
proteins, such as Fos and Jun and the ubiquitous transcription factor
SP1, are involved in ER-mediated transactivation of estrogen-responsive
genes that do not process the typical ERE (Gaub et al., 1990;
Wu-Peng et al., 1992; Krishnan et al., 1994;
Umayahara et al., 1994). Our preliminary data suggest that the
hERR1 binding element of the human lactoferrin gene did not bind AP1 or
SP1 (data not shown). At present, there is no evidence of ER
heterodimer with other receptors or transcription factors. Nonetheless,
several ER-associated proteins were recently identified (Halachmi et al., 1994; Cavailles et al., 1994, 1995). These
proteins bind to the estradiol-activated ER, but not to the inactive
ER. Whether hERR1 could interact with these ER-associated proteins
needs to be examined. It was interesting to find that hERR1 has no
effect on a strong palindromic ERE (Fig. 4C).
Therefore, hERR1 may not be a required coactivator for estrogen action,
but could be an integral part of estrogen response module specifically
for human lactoferrin gene. The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s) L38487 [GenBank](Genome Sequence Data Base).
Volume 271,
Number 10,
Issue of March 8, 1996 pp. 5795-5804
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)(for review, see Evans(1988) and Beato(1989)). In
many cases, other transcription factors bind near the HRE and interact
with the steroid hormone receptor to modulate the hormonal responses
(Bruggemeier et al., 1991; Danesch et al., 1987;
Espinas et al., 1994; Wieland et al., 1991; Zhang and
Young, 1991). For example, the direct participation of transcription
factors, SP1 and AP1, were recently found to modulate estrogen-induced
stimulation in several estrogen-responsive genes (Krishnan et
al., 1994; Umayahara et al., 1994; Wu-Peng et
al., 1992) lacking typical estrogen response elements (ERE)
(Klein-Hitpass et al., 1988, 1989). Thus, the hormonal
responsiveness of a particular gene is the result of a complicated
interplay between steroid receptors and other transcription factors.
Plasmids and Oligonucleotides
The pHL-414CAT
plasmid was constructed as described previously (Yang and Teng, 1994).
Location of the oligonucleotides used in EMSA are presented in Fig. 1A, and mutated nucleotides are marked in Fig. 3B. The oligonucleotides were synthesized on an
ABI 392 DNA/RNA synthesizer (Applied Biosystems, Foster City, CA) and
purified by column chromatography. The DNA sequences of
oligonucleotides containing BglII/BamHI linkers were
as follows: oligonucleotide -418/-378,
5`-ACCTGCCCTAACTGGCTCCTAGGCACCTTCAAGGTCATCTG-3`; M1,
5`-ACCTGCCCTAACTGGCTCCTAGGCACCTTCAACCTCATCTG-3`; M2,
5`-ACCTGCCCTAACTGGCTCCTACCCACCTTCAAGGTCATCTG-3`; M3,
5`-ACCTGCCCTAACTCCCTCCTAGGCACCTTCAAGGTCATCTG-3`; oligonucleotide
-375/-340, 5`- GAAGAAGATAGCAGTCTCACAGGTCAAGGCGATCTTG-3`.
The oligonucleotide containing the BglII linker was as
follows: oligonucleotide -418/-394,
5`-ACCTGCCCTAACTGGCTCCTAGGCAG-3`, oligonucleotide
-396/-382, 5`-GCACCTTCAAGGTCA-3`. Complement strands of the
above oligonucleotides were also synthesized and double-stranded DNA
prepared (Liu and Teng, 1992). An oligonucleotide corresponding to the
vitellogenin A2 ERE, 5`-GATCTAGGTCACAGTGACCTA-3` (Klein-Hitpass et
al., 1988), was also synthesized.
Mutagenesis
Mutations introduced into pHL-414CAT
were carried out by the transformer site-directed mutagenesis kit
(Clontech, Palo Alto, CA) according to the manufacturer's
instructions. The selected primer was
5`-GCAATTGTTGTTGATATCTTGTTTATTGCAGC-3`, and the mutated primers were as
follows: m1; 5`-GGCACCTTCAACCTCATCTGCTG-3`; m2;
5`-CTGGCTCCTACCCACCTTCAAGG-3`; m3;
5`-GCCCTAACTCCCTCCTAGGC-3`; m6,
5`-GATAGCAGTCTCACACCTCAAGGCGATCTTCA-3`; m7,
5`-GCAGTCTCACAGGTCAAGGTGACCTTCAAGTAAAGACCCTCTGC-3`.
Mutant d (mut d) was constructed by recombinant PCR (Higuchi, 1990).
The mutated primer was 5`-CTAGGCACCTTAAAGGTCATCTGC-3`. Plasmids
were selected, CsCl-purified, and sequenced with Sequenase version 2.0
kit (U. S. Biochemical Corp.) according to the specification of the
supplier. The mutated nucleotides were indicated in Fig. 3B, which is identical to the one made in EMSA.Nuclear Protein Preparation and EMSA
Human
endometrium carcinoma RL95-2 and mammary gland HBL 100 cells were grown
to an exponential stage and used for nuclear protein preparation as
described previously (Liu and Teng, 1992). EMSA was conducted as before
with minor modifications. Briefly, 1-2 µl of nuclear extract
(1-3 µg of protein) was mixed with 0.3-0.5 ng of
labeled probe (2-4 
10
cpm) in 10 µl of
reaction mixture containing 4 µg of poly(dI-dC), 10 mM HEPES, pH 7.9, 10% glycerol, 2% Ficoll-400, 40 mM NaCl,
and 2 mM dithiothreitol. The binding reaction was placed on
ice for 30 min, and the complexes were separated on a 3.5%
nondenaturing polyacrylamide gel (Yang and Teng, 1994).Methylation Interference Assay
The methylation
interference assay was conducted as described previously (Liu et
al., 1993). Briefly, a single-stranded FP1 oligonucleotide
(-418 to -378 plus BamHI/BglII cloning
site; Yang and Teng, 1994) was 3` end-labeled with T4 kinase and
[-
P]ATP, then annealed to the unlabeled
complementary strand. The double-stranded oligonucleotides were
gel-purified. The labeled DNA was incubated in a 200-µl reaction
mixture containing 50 mM dimethyl sulfate, 50 mM sodium cacodylate, and 1 mM EDTA, pH 8.0, at room
temperature for 3 min. DNA was purified by repeated ethanol
precipitation. About 5 ng of labeled and methylated DNA (4
10 
cpm) was incubated with 50 µg of nuclear protein
extract in a 60-µl reaction similarly to the EMSA. Free and
protein-DNA complexes were separated in a 3.5% nondenaturing gel. The
individual complexes and the free DNA were excised from the gel,
purified, and cleaved by 1 M piperidine at 90 °C for 30
min. The samples were extracted with phenol/chloroform, precipitated by
ethanol, and analyzed on an 8% sequencing gel. The G + A chemical
reaction for the same DNA fragment was included as a marker (Ausubel et al., 1990).Cell Culture, DNA Transfection, and Chloramphenicol
Acetyltransferase (CAT) Assay
Human endometrium carcinoma RL95-2
cells (ATCC CR1617) were grown in 1:1 mixture of Dulbecco's
minimal essential medium:Ham's F-12 supplemented with 10% fetal
bovine serum, 5 µg/ml of bovine insulin, and 100 units/ml
penicillin/streptomycin under 5% CO
. Transient
transfections were performed by the calcium phosphate method with a
Cellphect transfection kit (Pharmacia LKB Biotech). The CAT assays were
described previously (Liu and Teng, 1992), and the reaction products
were analyzed with an ascending TLC followed by quantitation using the
PhosphorImager System (Molecular Dynamic, Sunnyvale, CA). The cells
were cotransfected with vector alone or with 5 µg/well of the
reporter plasmid and 0.5 µg/well of the estrogen receptor
expression plasmid (HEO). After transfection, the cells were cultured
in 10% charcoal-stripped fetal bovine serum with or without hormone
(diethylstilbestrol, 10M) for 24 h. All
experiments were repeated at least three times with duplicated samples.
The results were presented as mean ± S.D.
Preparation of Probe for Expression cDNA Library
Screening
The 46 base pairs of oligonucleotide
(-418/-378, the FP1, plus BamHI/BglII
cloning sites) was multimerized with modifications in a direct head to
tail orientation according to Rosenfeld and Kelly(1986). After the
ligation reaction, the larger DNA fragments were isolated from 1.2%
agarose gel and subsequently subcloned into the BamHI/BglII sites of pSL1180 vector (Pharmacia). A
plasmid containing four tandem repeats of the FP1 sequence was
identified by sequencing and designated as pSLFP4. This plasmid was
used for duplication of the insert. The third round of duplication, we
obtained a plasmid, pSLFP32, containing 32 head to tail repeats of the
FP1 sequence. This DNA fragment was isolated from the plasmid,
purified, labeled with [
-P]dCTP, and used
as the probe for screening the expression cDNA library.
Construction and Screening of Expression cDNA
Libraries
Total RNA from log phase of RL95 cells was prepared by
the acid guanidinium thiocyanate-phenol-chloroform extraction method
(Chomczynski and Sacchi, 1987). The poly(A) RNA was
prepared by a standard oligo(dT) column method. The
gt22A
expression library was constructed with a cDNA synthesis kit according
to the manufacturer's specification (Life Technologies, Inc.).
The cDNA library contains 1.5
10
individual
recombinants.Generation of Prokaryotic Expression Recombinant
Plasmid
Larger amounts of -DNA from hERR1 recombinant were
prepared by standard methods (Maniatis et al., 1982). The cDNA
insert was recovered by NotI/SalI digestion and then
subcloned into the same restriction sites of the pGEX-4t-3 expression
vector (Pharmacia). The cloning site and the in-frame reading with
gt22A system were verified by dideoxy sequencing. This expression
recombinant was designated as pGEX-hERR1. The pGEX-hER was generated by
PCR the HEO plasmid with the following primers containing the EcoRI linker: the sense primer,
5`-GGGAATTCCATGACCATGACCCTCCACACCA-3` and the antisense primer,
5`-GGGAATTCTCAGACTGTGGCAGGGAAACCCT-3`. The PCR products were subcloned
into EcoRI site of the pGEX-4t-1.
Expression and Purification of Glutathione S-Transferase
Fusion Protein
The expression and purification of glutathione S-transferase fusion protein were carried out with RediPack
GST purification module according to the manufacturer's
specifications (Pharmacia). The fusion proteins, GST-hERR1 and GST-hER,
were prepared by batch method. Eluted proteins were analyzed on 10%
SDS-PAGE and visualized by Coomassie staining. After the protein
concentration was determined by Bio-Rad protein reaction, the protein
was aliquoted and stored at -70 °C until use.Polyclonal Antibody Production, Affinity Purification,
Western and Far-Western Analyses
A female New Zealand White
rabbit was immunized with 500 µg of purified GST-hERR1. The IgG
fraction was isolated by Sepharose chromatography and further purified
by affinity chromatography of the GST-hERR1 coupled to the Affi-Gel 10
(Bio-Rad) column.P-labeled GST-hERR1. To label the
protein, the Sepharose-bound GST-hERR1 was incubated with
[
-
P]ATP and cAMP-dependent protein kinase
(Sigma) in HMK buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 10 mM MgCl
) containing 1 mM dithiothreitol for 30 min. After washing, the P-GST-hERR1 was eluted from the Sepharose beads by reduced
glutathione buffer (10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0).
Northern Blot Analysis
Total RNA was extracted
(Chomczynski and Sacchi, 1987). The RNA samples were denatured in
formamide/formaldehyde, electrophoresed through a 1.2% agarose gel, and
transferred to a Hybond-N membrane (Amersham). An
oligonucleotide specific for the 5` end of the published hERR1 sequence
(180R, from nucleotide 1 to 180; Giguere et al., 1988) was
synthesized by PCR. The 5` oligonucleotide probe from hERR1 (185B, from
nucleotides 1 to 185, Fig. 6A) was obtained by cutting
the cDNA in clone FP1.4 with SalI/BstXI. The cDNA
probes,
-actin (Oncor, Gaithersburg, MD), 180R, and 185B were
radiolabeled to a specific activity of approximately 10
counts/min/µg with PRIME-IT 2 random primer labeling kit
(Stratagene, La Jolla, CA). Hybridization of the blots were described
previously (Liu and Teng, 1994).
-actin after
stripping. The position of 28 S, 18 S, hERR1 mRNA are indicated. C, detection of hERR1 by Western blotting. Proteins from
nuclear extract of the RL95-2, HBL100, HeLa, and Comma-D cells were
separated on a 10% SDS-PAGE and blotted onto the nitrocellulose. A
major 42-kDa protein and a minor 53-kDa protein (arrow) were
detected by antiserum to hERR1. The molecular markers are
indicated.
Mapping Nuclear Protein Binding Sites in FP1 Region of
Human Lactoferrin Promoter
Fig. 1A shows the
locations of oligonucleotides and the footprint areas of the human
lactoferrin gene. We examined the interactions of RL95-2 nuclear
proteins with the FP1 region (-418 to -378) by EMSA,
methylation interference, and mutagenesis. Nuclear extracts from RL95-2
cells mixed with radiolabeled FP1 oligonucleotide produced three more
slowly migrating complexes in a band shift assay and were designated
C1, C2, and C3 (Fig. 1B, lane 1). The competition
experiment demonstrated specific interactions for all three complexes (lane 2). In order to identify the DNA contacts for these
proteins, labeled FP1 was partially methylated before EMSA. The
individual bands were excised from the gel, and DNA methylation
interference analyses were performed. DNA contacts in the bottom strand
of DNA for all three complexes were obvious (Fig. 2A, lane
8, 9, and 10); however, only C1 and C3 contacts in the
top strand could be gleaned (Fig. 2A, lanes 5 and 3, respectively). The G contacts by the nuclear protein in all
three complexes were indicated in Fig. 2B.
Steroid Receptor ``Half-site'' in FP1 Region
Modulates Estrogen Responsiveness of the Human Lactoferrin
Gene
To examine whether the steroid receptor half-site in the
FP1 region plays any role in estrogen responsiveness, we constructed a
CAT reporter plasmid, pHL-414CAT, which contains 414 base pairs of the
human lactoferrin promoter/enhancer region. Both the imperfect ERE and
the FP1 were present. The wild type and mutated FP1 plasmids were
transfected together with estrogen receptor expression vectors into
human endometrial carcinoma RL95-2 cells. Fig. 4A
shows that the pHL-414CAT responded to estrogen stimulation with an
18-fold increase of CAT activity (Fig. 4A, wt). Mutations
made at all three locations in the FP1 region reduced the basal CAT
activity, hence the estrogen-stimulated activities accordingly (Fig. 4A, m1, m2, and m3). Nevertheless, the
folds of estrogen stimulation were maintained at m2 and m3.
Identifying the Critical Nucleotides in C1
Binding
To examine which nucleotides were involved in C1
binding, we used wild type and mutated oligonucleotides (-396 to
-362) to compete for binding in EMSA. The COUP-TF binding element
and the steroid receptor half-site were included in the 20-mer
double-stranded oligonucleotides. Fig. 5shows that every
nucleotide tested was important for C1 binding. Mutants c and d could
partially compete for binding at the C1 region (lanes
9-11). Mutation of C to A in mutant d did not affect COUP-TF
binding, since this nucleotide is at the center of the palindromic
COUP-TF binding element. Therefore, mutant d could compete with C2
complex (lanes 10 and 11) efficiently. Nucleotides
beyond the 3` end of the steroid receptor half-site were also needed
for C1 binding (data not shown). Thus, the nucleotide sequences at both
ends of the steroid receptor half-site were important for establishing
the C1 complex. Results from these studies suggest that the minimum C1
binding element is TCAAGGTCATC. Since m1 mutation prevents C1 complex
formation and hampers C2 binding (Fig. 3A, lane 2), it
is necessary to confirm that the protein in the C1 complex is actually
responsible for the enhanced estrogen-stimulated activity. We tested
mutant d (Fig. 5A) that binds C2 but not C1 in the
transfection assay. The results showed a reduction of estrogen
responsiveness similar to the m1 reporter construct (compare fold of
stimulation between m1 in Fig. 4and mutant d in Fig. 5B).
, control; &cjs2113;,
diethylstilbestrol.
Isolation and Identification of the cDNA Clone That Binds
to C1 Region
We screened an expression library made from poly(A)
RNA of the RL95-2 cells with a concatenated FP1 sequence in order to
isolate the nuclear protein that binds to the C1 region in human
endometrial cells. Among the 1.5 10 clones
screened, we identified six positive clones, and the longest, FP1.4,
was completely sequenced. From the nucleotide sequence of the cDNA and
the deduced amino acid sequence, we found that the FP1.4 was nearly
identical to hERR1 (Giguere et al., 1988). The major
differences between our cDNA and published hERR1 sequence occurred at
the 5` end and few internal deletions. To verify the cDNA sequence, we
submitted the FP1.4 clone to commercial sequencing (Lark Sequencing
Co.) and sequenced two additional clones isolated from the expression
cDNA library generated from our laboratory (RL95-2 cell), and two
clones obtained from the commercial expression cDNA library (human
hippocampus) at the regions in question (nucleotides 740-750 and
1190-1250) and confirmed the FP1.4 sequence. Among the 10 cDNA
clones isolated from both libraries, the FP1.4 was the longest, yet
still 176 base pairs less than the published hERR1 sequence at the 5`
end.Presence of Truncated hERR1 mRNA and Protein in the
RL95-2 Cells
By using 5` probes from the published hERR1
sequence (180R) and FP1.4 (185B), we examined the presence of hERR1
mRNA in RL95-2 cells and mouse kidney tissue. Fig. 6B shows that the 180R probe generated according to the published
hERR1 sequence (nucleotides 1-180; Giguere et al., 1988)
did not hybridize to any mRNA from RL95-2 cells (lane 1) and
mouse kidney tissue (lane 2), whereas the 185B probe from the
FP1.4 clone (nucleotides 1-185 in Fig. 6A)
detected a prominent hERR1 mRNA at the 2.2-kilobase region from both
samples (RL95-2 cells and mouse kidney tissue at lane 3 and 4, respectively). The same Northern blots were reprobed with
-actin as a positive control (Fig. 6B, lower
panel).Identification of the hERR1 as the C1-binding
Protein
The hERR1 fusion protein bound specifically to the FP1
oligonucleotides and produced a protein-DNA complex in EMSA (Fig. 7A, lane 1). The polyclonal antibody to hERR1
disrupted the protein-DNA complex (lane 3), whereas preimmune
serum did not (lane 2). By methylation interference analysis (Fig. 7B), we showed GST-hERR1 fusion protein expressed
in a bacterial system interacted with the TCAAGGTCATC element in FP1
region (lanes 3 and 8). To confirm that the hERR1 in
the RL95-2 nuclear protein indeed formed the C1 complex with FP1 in
EMSA, we incubated the hERR1 antibody with the RL95-2 nuclear protein
prior to the binding reaction. Fig. 8shows the three complexes
generated by the RL95-2 nuclear protein and FP1 interaction (lane
1). The hERR1 antibody, but not the preimmune serum, supershifted
only the C1 complex (lanes 2-4). To rule out the
possibility that the estrogen receptor also binds to the C1 region, we
used vitellogenin A2 (vit-A2) ERE in competition experiments. Even with
the inclusion of 100-fold molar excess of double-stranded vit-A2 ERE,
the oligonucleotides were unable to compete with C1 complex (Fig. 8, intensity of C1 in lanes 5-8). This
result was in agreement with our previous observations that the
estrogen receptor antibody (H222) did not interact with any of the
complexes (Yang and Teng, 1994).
hERR1 Interacts with ER through Direct Protein-Protein
Contacts
To examine whether hERR1 could interact with estrogen
receptor in vitro, we performed far Western analysis. Human ER
was expressed in the bacteria system as the GST fusion protein and
verified by Western blotting (data not shown). Fig. 9shows P-labeled hERR1 interacted with GST-ER fusion protein only (lanes 12 and 13). GST protein by itself (lane
11) nor with other GST fusion protein that were expressed
similarly interacted with the hERR1 (lanes 14-16).
P-labeled GST-hERR1 as
described under ``Materials and Methods.'' GST-HAV (viral
protein); GST-NS (mouse DNA-binding protein PO-GA, gift from T.
Sueyoshi); GST-EST (mouse testis estrogen sulfotransferase, gift from
W. C. Song); GST, protein alone; Alb, bovine serum albumin; ST,
standard; and GST-ER (human estrogen receptor). Lanes
1-8, Coomassie Blue stain. Lanes 9-16,
radiogram.
hKE4 and nucleotides 179-2,430 from
hKA1. It is possible that the RL95-2 cell and mouse kidney express
hERR1 mRNA with nucleotide sequence similar to the hKA1. Recent
evidence showed that multiple isoforms could be generated by members
from the steroid/thyroid receptor superfamily through different
promoter usage and alternative RNA splicing (Ikeda et al.,
1993; Giguere et al., 1994; Guiramand et al., 1995).
The same mechanism could be used to produce different forms of hERR1 in
various cell types or tissues. We cannot exclude the possibility that
hKE4 sequence was present in a minor portion of the hERR1 mRNA in
RL95-2 cells and mouse kidney, however, undetectable by the limited
sensitivity of Northern blot analysis. Consistent with the short hERR1
mRNA in the RL95-2 cells, the major nuclear protein detected by hERR1
antibody in Western blot was 42 kDa. Therefore, hERR1 in the RL95-2
cells might be translated from the Met at nucleotide 177 (Fig. 6A), which predicated a 47-kDa protein. A minor
53-kDa protein was also detected by the hERR1 antibody in the nuclear
extract of RL95-2 and HeLa cells (Fig. 6C).
Posttranslational modification and degradation might produce a protein
larger or smaller than predicated size from its amino acid sequence. It
has been reported that hERR1 was copurified with COUP-TF as 53 kDa
(Wang et al., 1991) and with a cellular transcriptional
repressor of the SV40 major late promoter as 55 kDa (Wiley et
al., 1993) protein from HeLa cell nuclear extract. Whether these
hERR1 proteins were encoded by the same hERR1 mRNA in the RL95-2 cells
is unknown. Reverse transcriptase PCR of various human tissue and cell
line RNAs with specific hERR1 primers might reveal different forms of
hERR1 mRNA. Alternatively, different hERR1 proteins could be detected
by antibodies generated to specific peptides at different regions of
the hERR1.
)Sequence comparison between human and mouse ERR1 revealed
that the homologies are 90% in nucleotides and 98% in amino acid. This
finding suggests that the hERR1 is evolutionary conserved. Protein
alignment and dendogram analysis of the hERR1 to other steroid
receptors show a close relationship to ER, particularly the DNA binding
domain. There is 68% homology at this region and the nine cystine
residues constituting the zinc-fingers are conserved (Green et
al., 1986). This is paradoxical, since the hERR1 binds an extended
AGGTCA motif and ER binds palindromic AGGTCA as dimer (see review by
Glass(1994) and references therein). The mutagenesis and EMSA
competition experiments (Fig. 5) suggest that the nucleotides
surrounding the AGGTCA are important in order for hERR1 to bind. It is
likely that the hERR1 belongs to the new subclass of orphan receptors
(Ueda and Hirose, 1990; Wilson et al., 1992; Lavorgna et
al., 1991; Tsukiyama and Niwa, 1992; Ikeda et al., 1993;
Giguere et al., 1994) that bind to the extended steroid
receptor half-site as a monomer (Wilson et al., 1993).
))
We thank P. Chambon of CNRS of France for kindly
providing the human estrogen receptor expression plasmid and S. Tsai of
Baylor College of Medicine for the COUP-TF antibody, W. C. Song and T.
Sueyoshi of NIEHS for GST fusion proteins, M. Kricker of ISN for
computer analysis, C. Weinberger and M. Negishi for critical reading of
the manuscript, and L. Belans for editorial assistance.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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