A cytoplasmic oestrogen-binding component in chicken liver.

A macromolecular component in the liver cytosol from laying hens as well as roosters, protein in nature and sedimenting at 4S, was shown to bind oestradiol. The dissociation constant (Kd) of the complex is approximately 5 X 10(-6)M. No binding component with a higher affinity for oestradiol was detectable in the cytosol. The binding is specific for the tissue and hormone, with the exception that progesterone also shows some affinity for this 4S component. The number of binding sites is about 330 pmol/mg cytosol protein. This number is not altered significantly after treatment of a rooster with oestrogen (24 h) or with cycloheximide (3 h). The cytoplasmic complex (oestradiol-4S-component) does not enhance the binding of oestradiol to the chromatin from rooster liver. The nuclear complex (oestradiol bound to the soluble nuclear receptor seems to be more effective in doing so.     

Protein-DNA interactions in vitro with 5'-flanking DNA fragments from the chicken vitellogenin gene

The expression of the vitellogenin gene in the liver of oviparous animals is under strict control of estrogen. We have studied the interaction of proteins extracted from nuclei of different estrogen responsive tissues with two fragments (-728 to -470 and -625 to -470) of the upstream region of the chicken vitellogenin gene, using the gel-retardation technique. We found a complex pattern of retarded bands using nuclear extracts from laying hen liver, rooster liver and MCF-7 cells. The patterns observed display differences in the position and intensities of some of the bands, depending on the source of the extract used. The possible significance of these findings will be discussed.     

Binding of type II nuclear receptors and estrogen receptor to full and half-site estrogen response elements in vitro.

The mechanism by which retinoids, thyroid hormone (T3) and estrogens modulate the growth of breast cancer cells is unclear. Since nuclear type II nuclear receptors, including retinoic acid receptor (RAR), retinoid X receptor (RXR) and thyroid hormone receptor (TR), bind direct repeats (DR) of the estrogen response elements (ERE) half-site (5'-AGGTCA-3'), we examined the ability of estrogen receptor (ER) versus type II nuclear receptors, i.e. RARalpha, beta and gamma, RXRbeta, TRalpha and TRbeta, to bind various EREs in vitro . ER bound a consensus ERE, containing a perfectly palindromic 17 bp inverted repeat (IR), as a homodimer. In contrast, ER did not bind to a single ERE half-site. Likewise, ER did not bind two tandem (38 bp apart) half-sites, but low ER binding was detected to three tandem copies of the same half-site. RARalpha,beta or gamma bound both ERE and half-site constructs as a homodimer. RXRbeta did not bind full or half-site EREs, nor did RXRbeta enhance RARalpha binding to a full ERE. However, RARalpha and RXRbeta bound a half-site ERE cooperatively forming a dimeric complex. The RARalpha-RXRbeta heterodimer bound the Xenopus vitellogenin B1 estrogen responsive unit, with two non-consensus EREs, with higher affinity than one or two copies of the full or half-site ERE. Both TRalpha and TRbeta bound the full and the half-site ERE as monomers and homodimers and cooperatively as heterodimers with RXRbeta. We suggest that the cellular concentrations of nuclear receptors and their ligands, and the nature of the ERE or half-site sequence and those of its flanking sequences determine the occupation of EREs in estrogen-regulated genes in vivo .     

Isolation and characterization of a cDNA clone specific for avian vitellogenin II.

A clone for vitellogenin, a major avian, estrogen responsive egg yolk protein, was isolated from the cDNA library of estrogen-induced rooster liver. Two forms of plasma vitellogenin, vitellogenin I (VTG I) and vitellogenin II (VTG II), distinguishable on the basis of their unique partial proteolysis maps, have been characterized and their corresponding hepatic precursor forms identified. We have used this criterion to specifically characterize which vitellogenin protein had been cloned. Partial proteolysis maps of BTG I and VTG II standards, synthesized in vivo, were compared to maps of protein synthesized in vitro using RNA hybrid-selected by the vitellogenin plasmid. Eight major digest fragments were found common to the in vitro synthesized vitellogenin and the VTG II standard while no fragments were observed to correspond to the VTG I map. A restriction map of the VTG II cDNA clone permits comparison to previously described cDNA and genomic vitellogenin clones.     

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)     

Estradiol-dependent trans-acting factor binds preferentially to a dyad-symmetry structure within the third intron of the avian vitellogenin gene

The secondary activation of the avian vitellogenin II gene in isolated liver nuclei by cytoplasmatic liver extracts of estradiol-treated chicks is accompanied by the binding of a protein from the extract to the structural part of the cloned gene. Both the DNA-binding and gene-stimulatory activities, which cochromatograph on heparin-Sepharose, are apparently present only in the cytoplasmatic liver extracts of estradiol-treated roosters and in the oviduct extracts of egg-laying hens. DNA-binding competition assays combined with exonuclease III footprinting showed that the factor binds to the imperfect dyad-symmetry structure 5'GTCTTGTTCCAAAC3' within the third intron of the gene. The factor is sequence specific and binds equally well to both single-and double-stranded DNA with an estimated dissociation constant of 3.5 X 10(-10) M.     

Association of transcriptionally active vitellogenin II gene with the nuclear matrix of chicken liver

Supercoiled DNA loops linked to the nuclear matrix can be progressively cleaved with deoxyribonuclease I. The DNA which remains associated with the nuclear matrix can be purified and analysed for vitellogenin II sequence content by dot blot hybridization. Using this technique we show that vitellogenin II gene sequences are selectively associated with the nuclear matrix of liver but not with oviduct of laying hens. Following primary stimulation in immature chicks of vitellogenin synthesis with estradiol, the association of the gene with the nuclear matrix precedes vitellogenin mRNA synthesis. After 15 days when the level of vitellogenin mRNA has returned to zero, the gene is no longer preferentially associated with the nuclear matrix. At this time a second stimulation with estradiol results in a reassociation of the vitellogenin II gene with the nuclear matrix. In addition to the structural gene, both the 3' and 5' end flanking regions (1.5-2 kb) also bind to the nuclear matrix. However, beyond the limit of 1.5-2 kb upstream from the 5' end of the gene, there is no preferential binding of DNA to the nuclear matrix.     

An estrogen-inducible protein binds specifically to a sequence in the 3'' untranslated region of estrogen-stabilized vitellogenin mRNA.

The 3' untranslated region (3'-UTR) has been implicated in the estrogen stabilization of hepatic Xenopus laevis vitellogenin mRNA. We used RNA gel mobility shift assays to demonstrate that Xenopus liver contains a factor which binds with very high specificity to a segment of the 3'-UTR of vitellogenin B1 and B2 mRNAs. We detected a single high-affinity binding site in the vitellogenin mRNA 3'-UTR and localized the binding site to a 27-nucleotide region. Since binding was abolished by proteinase K digestion, at least a component of the factor is a protein. Following estrogen administration, binding was induced approximately four- to fivefold in extracts from liver polysomes. The hepatic vitellogenin mRNA-binding protein was found in both polysomes and cytosol. Since the protein was also estrogen inducible in cytosol, this represents a genuine induction, not simply recruitment of the cytosolic protein into polysomes. UV cross-linking studies with the 27-nucleotide recognition sequence revealed bands corresponding to bound proteins with apparent molecular weights of 71,000 and 141,000. This appears to be the first example of steroid hormone-inducible proteins binding to an mRNA 3'-UTR. Its induction by estrogen and its sequence-specific binding to a region of vitellogenin mRNA important in estrogen-mediated stabilization suggest that the protein may play a role in the regulation of mRNA stability.     

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.