Sp1 binding sites and an estrogen response element half-site are involved in regulation of the human progesterone receptor A promoter

Progesterone receptor gene expression is induced by estrogen in MCF-7 human breast cancer cells. Although it is generally thought that estrogen responsiveness is mediated through estrogen response elements (EREs), the progesterone receptor gene lacks an identifiable ERE. The progesterone receptor A promoter does, however, contain a half-ERE/Sp1 binding site comprised of an ERE half-site upstream of two Sp1 binding sites. We have used in vivo deoxyribonuclease I (DNase I) footprinting to demonstrate that the half-ERE/Sp1 binding site is more protected when MCF-7 cells are treated with estrogen than when cells are not exposed to hormone, suggesting that this region is involved in estrogen-regulated gene expression. The ability of the half-ERE/Sp1 binding site to confer estrogen responsiveness to a simple heterologous promoter was confirmed in transient cotransfection assays. In vitro DNase I footprinting and gel mobility shift assays demonstrated that Sp1 present in MCF-7 nuclear extracts and purified Sp1 protein bound to the two Sp1 sites and that the estrogen receptor enhanced Sp1 binding. In addition to its effects on Sp1 binding, the estrogen receptor also bound directly to the ERE half-site. Taken together, these findings suggest that the estrogen receptor and Sp1 play a role in activation of the human progesterone receptor A promoter.     

Hormonal regulation of vitellogenin genes: an estrogen-responsive element in the Xenopus A2 gene and a multihormonal regulatory region in the chicken II gene

Expression of the vitellogenin genes in avian and amphibian liver is regulated by estrogens. The DNA elements mediating estrogen induction of the various vitellogenin genes of chicken and Xenopus encompass one or more copies of a 13-mer palindromic sequence called the estrogen-responsive element (ERE). Here we show that upon incubation with the purified estrogen receptor (ER) from calf uterus the Xenopus vitellogenin A2 gene yields a DNase-I footprint over the ERE between -331 and -319. This element does not mediate the response to glucocorticoids or progestins in T47D cells. The three guanine residues in each half of the palindrome are protected against methylation by dimethylsulfate after incubation with ER, but not with glucocorticoid (GR) or progesterone (PR) receptors. In contrast, the chicken vitellogenin II gene exhibits multihormonal regulation by estrogens, progestins, and glucocorticoids in T47D and MCF7 cells. Regulation is mediated by the DNA region between -721 and -591 that contains four binding sites for hormone receptors, as demonstrated by DNase-I footprints and methylation protection experiments. The two distal and most proximal binding sites are recognized by ER, GR, and PR, whereas the central binding site is only bound by ER and GR. At suboptimal concentrations, estrogens and progestins or glucocorticoids act synergistically. In experiments using a DNA fragment containing an ERE adjacent to a glucocorticoid-responsive element/progesterone-responsive element, ER and PR bind synergistically to their corresponding sites, perhaps explaining the functional synergism of both hormones. Thus, two very different regulatory elements are used to mediate estrogen induction of related genes in chickens and amphibians.     

Specific binding of estrogen receptor to the estrogen response element.

Gene transfer studies have shown that estrogen regulation of specific genes is mediated by estrogen response elements (ERE). We report that binding of the estrogen receptor to the ERE can be detected by a gel retardation (band shift) assay. This binding interaction was highly sequence and receptor specific. Methylation interference analysis showed that the ERE contact sites of estrogen receptor displayed a perfect twofold rotational symmetry. This is compatible with estrogen receptor binding to the ERE as a head-to-head dimer.     

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.     

Transcriptional regulation of the mouse calbindin-D9k gene by the ovarian sex hormone

Calbindin-D9k levels in the rat uterus are under the control of estrogen. We found that the putative estrogen response element (ERE) failed to bind to the estrogen receptor from the mouse uterus. We therefore isolated mouse genomic clones of the calbindin-D9K gene and analyzed their expression in the mouse uterus. The promoter region of the gene contained several putative steroid hormone receptor binding sites. To characterize these elements, we constructed several promoter-reporter plasmids, and transiently transfected them into T47D breast cancer cells that express both estrogen and progesterone receptors. Luciferase activity was expressed from a promoter region containing the putative progesterone response element (PRE) and expression was stimulated by progesterone. In the uterus of oophorectomized mice, the calbindin-D9k gene was up-regulated by progesterone, but not by estrogen. These results suggest that the mouse uterine calbindin-D9k gene is expressed under the control of a PRE.     

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.     

Progesterone induction of metallothionein-IIA gene expression

Expression of the metallothionein gene is known to be induced by glucocorticoids in a variety of cells. Here we show that in human cell lines containing functional progesterone receptors, the endogenous metallothionein-IIA (hMTIIA) gene is inducible by the synthetic progestins R5020 and medroxy-progesterone acetate. That this effect reflects a direct interaction with the metallothionein gene is supported by our finding that the partially purified progesterone receptor binds to the promoter region of the gene in vitro. The limits of the DNase I footprint and the guanine residues protected in methylation studies with the progesterone receptor are similar to those previously described for the glucocorticoid receptor. Thus, the hormone regulatory element of the human metallothionein-IIA gene can mediate regulation by both glucocorticoids and progestins, as does the hormone regulatory element of mouse mammary tumor virus.     

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.