Synergism of closely adjacent estrogen-responsive elements increases their regulatory potential

Recent gene transfer experiments have shown that an estrogen-responsive DNA element (ERE) GGTCANNNTGACC mediates the estrogen inducibility of the Xenopus laevis vitellogenin A1 and A2 genes as well as the chicken vitellogenin II gene. We report here on experiments that explain the estrogen regulation of the Xenopus vitellogenin B1 and B2 genes. In these genes, two ERE homologues, which have only low, if any, regulatory capacity on their own, act synergistically to achieve high estrogen inducibility. Furthermore we show that synergism of EREs is most efficient, when the two elements are closely adjacent and that it is lost when the synergistic elements are separated by 125 basepairs. In-vitro estrogen receptor binding experiments indicate that co-operative binding of estrogen receptors to closely adjacent EREs is not essential for synergism of ERE homologues that have no intrinsic regulatory capacity. Functional synergism of EREs is observed in the human estrogen-responsive MCF-7 cell line as well as in mouse fibroblasts (Ltk-) cotransfected with estrogen receptor expression vectors. Even expression of a truncated receptor protein lacking 178 amino acid residues of the amino-terminal end allows synergism, suggesting that the amino-terminal end preceding the DNA-binding domain of the estrogen receptor is not required.     

Interaction of estrogen receptor complexes with the promoter region of genes that are negatively regulated by estrogens: the alpha 2u-globulins.

Since estrogens strongly suppress the expression of alpha 2u-globulin genes in the rat liver, we studied the binding of estrogen-receptor complexes to fragments derived from alpha 2u-globulin gene RAO 01 using a DNA-cellulose competition assay. Rat uterus cytosol labelled with [3H]estradiol was used as a source of the estrogen receptor. As a positive control in these experiments we used an oligonucleotide containing the estrogen response element (ERE) cloned into pUC18. Our experiments indicate that estrogen-receptor complexes bind specifically to the ERE and to a fragment of RAO 01 located in the 5'-upstream region (bp -606 up to -575). This fragment is conserved among other members of this gene family. This is the first time that in vitro estrogen receptor binding is observed to gene fragments derived from a gene that is repressed by this steroid in vivo.     

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.