The hormone regulatory element of mouse mammary tumour virus mediates progesterone induction.

Sequences within the long terminal repeat region (LTR) of mouse mammary tumour virus (MMTV) confer progestin inducibility to either the tk-promoter or the MMTV-promoter in T47D cells, a human mammary tumour cell line which possesses high constitutive levels of progesterone receptor. In a clone of MCF7 cells, another human mammary tumour cell line with a low level of progesterone receptor, as well as in rat fibroblasts, glucocorticoid but not progestin induction is observed. The effect of the progesterone analogue R5020 is much more pronounced than the effect of dexamethasone, and at the concentrations required for maximal induction, R5020 does not significantly compete with binding of dexamethasone to the glucocorticoid receptor. In conjunction with previous results on the DNA binding of the glucocorticoid and progesterone receptors, these data show that two different steroid hormones, acting through their respective receptors, can mediate the induction of gene expression by interacting with the same DNA sequences. Our results suggest that the hormone regulatory element of MMTV may primarily be a progesterone-responsive element in mammary cells.     

Molecular interactions of steroid hormone receptor with its enhancer element: evidence for receptor dimer formation

A steroid hormone responsive element (GRE/PRE), sufficient to confer glucocorticoid and progesterone inducibility when linked to a reporter gene, was used in band-shift assays to examine its molecular interactions with steroid hormone receptors. Both progesterone and glucocorticoid receptors bound directly and specifically to the GRE/PRE. The purine contact sites for both form A and form B chicken progesterone receptor, as well as those for rat glucocorticoid receptor, are identical. A peptide fragment produced in bacteria that primarily contain the DNA binding domain of the glucocorticoid receptor binds first to the TGTTCT half-site of the GRE/PRE, and a second molecule binds subsequently to the TGTACA (half-site) of the GRE/PRE in a cooperative manner. Utilizing the peptide fragment and the protein A-linked fragment, we demonstrated that the receptor interacts with its cognate enhancer as a dimer.     

Identification of two distinct nuclear factors with DNA binding activity within the glucocorticoid regulatory region of the rat alpha-1-acid glycoprotein promoter

Efficient glucocorticoid induction of alpha-1-acid glycoprotein (AGP) mRNA in rat hepatoma cells HTC (JZ-1) requires the activity of one or more preexisting and labile proteins acting cooperatively with the glucocorticoid receptor. Inhibiting protein synthesis markedly diminishes the glucocorticoid induction of rat AGP mRNA without affecting the inducibility of other glucocorticoid inducible genes such as the mouse mammary tumor virus (MMTV) or tyrosine amino transferase (TAT). The sequences responsible for conferring glucocorticoid inducibility to the rat AGP gene have localized on the AGP promoter between nucleotides -121 and -42. A typical glucocorticoid regulatory element (GRE) is found between residues -121 and -105 and downstream of this are the sequences (-90 to -42) responsible for the cycloheximide inhibition of the hormonal induction (10). Using mobility shift assays we have characterized the binding of two proteins or complexes of proteins to this promoter region (-90 to -64). Our data show that the binding of these factors (called ANF-1 and ANF-2) to the DNA is highly specific, and is not directly affected by cycloheximide. Furthermore a second binding site for ANF-2 has been localized in the AGP regulatory region to a sequence that overlaps the GRE.     

Differential gene activation by glucocorticoids and progestins through the hormone regulatory element of mouse mammary tumor virus

The hormone regulatory element (HRE) of mouse mammary tumor virus can mediate activation of an adjacent promoter by glucocorticoids and progestins. A detailed comparison of the DNA binding of receptors for both hormones using DNAase I footprinting and methylation protection detects clear differences in their interactions with the HRE region between positions -130 and -100. Binding studies and gene transfer experiments with a variety of mutants covering the entire HRE demonstrate differences in the relevance of the individual sequence motifs for induction by each hormone. The influence of changes in the angular orientation of receptor binding sites is also different for glucocorticoid and progesterone induction. In transfection experiments with mutated HREs, we find a functional cooperation between the receptor binding sites that does not correlate with variations in the in vitro affinity of the receptors for the corresponding DNA fragment.     

Functional interaction of hybrid response elements with wild-type and mutant steroid hormone receptors.

Steroid hormone receptors can be divided into two subfamilies according to the structure of their DNA binding domains and the nucleotide sequences which they recognize. The glucocorticoid receptor and the progesterone receptor (PR) recognize an imperfect palindrome (glucocorticoid responsive element/progesterone responsive element [GRE/PRE]) with the conserved half-sequence TGTYCY, whereas the estrogen receptor (ER) recognizes a palindrome (estrogen responsive element) with the half-sequence TGACC. A series of symmetric and asymmetric variants of these hormone responsive elements (HREs) have been tested for receptor binding and for the ability to mediate induction in vivo. High-resolution analysis demonstrates that the overall number and distribution of contacts with the N-7 position of guanines and with the phosphate backbone of various HREs are quite similar for PR and ER. However, PR and glucocorticoid receptor, but not ER, are able to contact the 5'-methyl group of thymines found in position 3 of HREs, as shown by potassium permanganate interference. The ER mutant HE84, which contains a single amino acid exchange, Glu-203 to Gly, in the knuckle of ER, creates a promiscuous ER that is able to bind to GRE/PREs by contacting this thymine. Elements with the sequence GGTCAcagTGTYCT that represent hybrids between an estrogen response element and a GRE/PRE respond to estrogens, glucocorticoids, and progestins in vivo and bind all three wild-type receptors in vitro. These hybrid HREs could serve to confer promiscuous gene regulation.     

Glucocorticoid-receptor interaction and induction of murine mammary tumor virus.

The relationship between the cellular uptake of glucocorticoid hormones, the binding of these hormones to specific in vitro receptors, and the induction of mouse mammary tumor viruses in an established mouse mammary tumor cell line was highly correlated. These results suggest that the induction of mouse mammary tumor virus by glucocorticoid hormones is a physiological process acting through a mechanism of high affinity, saturable steroid-receptors. A temperature-sensitive or salt-dependent step following glucocorticoid-receptor interaction was required for nuclear uptake of the steroid. Induction studies with different adrenocorticoids indicate that the synthetic glucocorticoid, dexamethasone (1,4-pregnadiene-9-fluor-16alpha-methyl-11beta,17alpha,21-triol-3,20-dione), is the most potent inducer of mouse mammary tumor viruses and all steroids which caused significant induction were glucocorticoids. Other glucocorticoids appear to stimulate murine mammary tumor virus production by a mechanism similar to that of dexamethasone; for example, corticosterone competes with dexamethasone for binding to the glucocorticoid receptor and blocks the uptake of dexamethasone into cells. Progesterone also blocks the cellular uptake of dexamethasone and can bind to the glucocorticoid receptor at low concentrations (10-7 to 10-8 M) but progesterone does not consistently induce virus at hormone concentrations even as high as 10-4 M. Thus, in this system, binding to a cytoplasmic receptor is necessary but not sufficient for induction by glucocorticoids. Estrogens and androgens interfere with receptor binding and cellular uptake of dexamethasone but only at much higher concentration (10-4 M) than progesterone, and do not induce mammary tumor virus production. Although there was a positive correlation between steroid structure, binding, and biologic induction, other factors clearly affect the physiological manifestations of steroid actions. Mouse cells with comparable cytoplasmic receptor levels and comparable nuclear uptake differed absolutely in their degree of murine mammary tumor virus induction following hormone treatment. Although all mouse cells examined contain comparable levels of murine mammary tumor virus DNA, only cells producing constitutive levels of murine mammary tumor virus RNA could be induced to higher levels by a variety of glucocorticoids.     

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.