An estrogen-responsive element derived from the 5' flanking region of the Xenopus vitellogenin A2 gene functions in transfected human cells

In the human breast cancer cell line MCF-7, we observe estrogen induction of the stable transfected Xenopus vitellogenin A2 gene. An estrogen-responsive element (ERE) could be defined by using a vitellogenin-chloramphenicol acetyltransferase hybrid gene in transient transfection experiments. The ERE is located in the 5' flanking region and is able to confer estrogen inducibility to the thymidine kinase gene promoter. By 5' and 3' deletions we have determined a 35 bp sequence sufficient for high stimulation by estradiol. Even 18 bp give a small estrogen response. The 35 bp ERE contains the palindromic sequence 5'GGTCACAGTGACC-3' as an essential element. The fact that the ERE of a frog gene functions in human cells demonstrates that signals and factors involved in the control have been conserved during evolution.     

Uterine estrogen receptor-DNA complexes: effects of different ERE sequences, ligands, and receptor forms

Previous studies used the gel retardation assay to examine the binding of the mouse estrogen receptor (ER) to the estrogen-responsive element (ERE) from the vitellogenin A2 gene (VitA2ERE). Multiple specific complexes were formed when the ER was bound to various estrogen agonists or antagonists, or in the absence of bound hormone. The ERE from the human PS2 gene, which varies from the consensus ERE by one base change in the right arm, was used in this study to determine the effect of DNA sequence on ER-ERE interaction with various ligand-receptor complexes. Partially purified ligand-free soluble ER showed a 3-fold lower affinity for the PS2ERE than for the VitA2ERE, suggesting a possible influence of the imperfect DNA sequence on certain binding interactions. However, multiple complexes of similar affinity were formed with the PS2 sequence by nuclear ER regardless of the agonist or antagonist bound. In gel retardation experiments, antagonist (LY117018) nuclear ER complexes bound to either PS2 or VitA2ERE migrated more slowly than agonist complexes, indicating that the slower migrating form of the complex was not due to the DNA sequence. Interestingly, soluble ER bound by LY 117018 did not produce this decreased mobility complex, suggesting that it was specific to the nuclear form of the ER antagonist complex. Receptor activation has been linked with exposure to increased temperature, resulting in an ER form that has an increased affinity for DNA. The binding of molybdate-stabilized nonactivated 8S ER to VitA2ERE was studied to determine the effect of temperature on ER binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

A far upstream estrogen response element of the ovalbumin gene contains several half-palindromic 5'-TGACC-3' motifs acting synergistically.

We have identified an estrogen-responsive enhancer element (DH3 ERE) in the estrogen-induced DNAase I-hypersensitive region III of the chicken ovalbumin gene, which is located approximately 3.3 kb upstream from the mRNA start site and does not contain palindromic ERE. Four TGACC half-palindromic motifs, separated from each other by more than 100 bp, are responsible for conferring estrogen inducibility either to the proximal ovalbumin gene promoter or to heterologous promoters. Thus, widely spaced half-palindromic ERE motifs can act synergistically. Each half-palindromic motif was shown to bind the estrogen receptor (ER) with a low efficiency in vitro. However, two widely spaced half-palindromic motifs bound the ER cooperatively, much more efficiently than expected from binding to isolated half-ERE motifs. The ovalbumin promoter half-palindromic ERE motif located close to the TATA box was required for the activity of the distal DH3 ERE, but could be replaced by the binding sites of other transactivators.     

Two functional estrogen response elements are located upstream of the major chicken vitellogenin gene.

We used a transient-expression assay to identify two estrogen response elements (EREs) associated with the major chicken vitellogenin gene (VTGII). Each element was characterized by its ability to confer estrogen responsiveness when cloned in either orientation next to a chimeric reporter gene consisting of the herpes simplex virus thymidine kinase promoter and the chloramphenicol acetyl transferase-coding region. Deletion analyses indicated that sequences necessary for the distal ERE resided within the region from -626 to -613 (nucleotide positions relative to the VTGII start site) whereas those necessary for the proximal ERE were within the region from -358 to -335. These distal and proximal elements contain, respectively, a perfect copy and an imperfect copy of the 13-base-pair sequence that is an essential feature of the EREs associated with two frog vitellogenin genes. These chicken VTGII EREs mapped near regions that were restructured at the chromatin level when the endogenous VTGII gene was expressed in the liver in response to estradiol. These data suggest a model for the tissue-specific expression of this estrogen-responsive gene.     

A common ancestor DNA motif for invertebrate and vertebrate hormone response elements.

The ecdysone-responsive DNA sequence of the Drosophila hsp27 gene promoter contains four direct and inverted repeats reminiscent of those that compose the vertebrate palindromic estrogen response element (ERE) and the thyroid hormone/retinoic acid response element (TRE/RRE). Interestingly, a 3 bp substitution in the wild-type Hsp27 ecdysone response element (EcdRE) increases both its similarity with the vertebrate ERE and TRE/RRE and its capacity to confer ecdysone responsiveness to a heterologous promoter. Remarkably, increasing the spacing between the inverted repeats of this strong EcdRE by two nucleotides converts it into an ERE. Inversely, decreasing the spacing between the two inverted repeats of the vertebrate consensus palindromic ERE, from three to one nucleotide, converts it into a functional EcdRE. Thus, the only difference between an invertebrate EcdRE and a vertebrate palindromic ERE or TRE/RRE is in the spacing between the conserved inverted repeated motifs forming these palindromic HREs. The finding that the sequence motif 5'-GGTCA-3' present in the vertebrate ERE and TRE/RRE is also a functionally important characteristic of an invertebrate HRE, suggests that a common ancestor regulatory DNA sequence gave rise to all HREs known so far. We discuss the possibility that this progenitor motif is the GGTCA sequence.     

Estrogen receptor alpha interaction with estrogen response element half-sites from the rat prolactin gene

Estrogen regulation of the rat prolactin gene requires sequences within the DNase I hypersensitive site II (HSII). We have used overexpressed mouse estrogen receptor alpha (ERalpha) protein to study interactions of ERalpha with an imperfect estrogen response element (ERE) and four ERE half-site sequences from HSII. We confirmed that ERalpha has higher affinity for ERE half-sites than for the imperfect ERE. As expected, the imperfect ERE formed a complex with ERalpha similar to that between mERalpha and a consensus ERE in gel shift assays. The ERalpha complex with half-sites, however, had faster mobility on a 4% polyacrylamide gel than the ERalpha complex with a consensus ERE, indicating that the complexes had different compositions. Ferguson analysis revealed that the ERalpha/half-site complex had a larger molecular weight and higher negative charge than the ERalpha/consensus ERE complex. Similar results were observed with purified human ERalpha, showing that the ERalpha/half-site complex contained only ERalpha and oligonucleotides. These results are best explained by a model in which a dimer of ERalpha is bound to two half-site oligonucleotides. We propose that two ERalpha dimers may interact with the four ERE half-sites in HSII to influence estrogen regulation of this gene.     

In vitro binding of the purified hormone-binding subunit of the estrogen receptor to oligonucleotides containing natural or modified sequences of an estrogen-responsive element

Estrogen receptor (ER) was purified from calf uterus by immunoaffinity chromatography in the absence of the ligand. The purified ER consists of a mixture of monomer and homodimer forms of 67-kDa hormone-binding subunit (no 90-kDa heat shock protein is present). The purified ER was incubated with a 32P-labeled 61-basepair oligonucleotide containing the sequence of the estrogen response element (ERE) of the Xenopus laevis A2 vitellogenin gene. DNA mobility shift assays showed formation of specific complexes of the ERE containing oligonucleotide with ER, formation which did not require and was not affected by estradiol or antiestrogenic molecules. Both the monomer and the dimer were equally able to interact with the ERE-containing oligonucleotide. Sucrose gradient experiments showed that only the ER monomer is able to interact with an oligonucleotide in which a single mutation destroyed the dyad symmetry of ERE. Multiple symmetric mutations which did not alter the dyad symmetry of ERE nevertheless totally destroyed the ability of the oligonucleotide to form complexes with either the monomeric or dimeric form of ER. These results suggest that ER is able to bind to ERE independently of the presence of estradiol or other proteins and, therefore, that estradiol does not act by modulating the ability of ER to bind to ERE on DNA.