(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) ()(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.
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 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).
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 -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.
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. , control; &cjs2113;,
diethylstilbestrol.
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 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.
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 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.
By using hERR1 as a probe, we isolated several cDNA
clones from mouse brain and kidney cDNA libraries. ()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).
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).