COUP-TFI is a potential regulator of retinoic acid-modulated development in Xenopus embryos.

Isomers of retinoic acid are considered likely regulators of developmental pattern formation in vertebrate embryos. The orphan receptor COUP-TFI, which can alter cellular responses to retinoic acid in cultured cells, is expressed in distinct regions of the developing zebrafish and mouse anterior central nervous system. We asked if COUP-TFI can modulate retinoic acid signaling and anterior neural development in a vertebrate embryo by examining: (1) whether COUP-TFI could alter transcriptional responses to retinoic acid in Xenopus embryonic explants, and (2) whether misexpression of COUP-TFI could regulate anterior neural gene expression during early Xenopus development. The results from these studies show that COUP-TFI is a potent regulator of retinoic acid-induced gene expression in Xenopus embryonic cells, and that misexpression of COUP-TFI causes deficiencies in anterior neural structures and head development in Xenopus embryos with a concomitant change in anterior neural gene expression. These results support the proposition that COUP-TFI has a role in the elaboration and patterning of anterior neural gene expression in vertebrates, possibly via effects on the retinoic acid signaling pathways.

Estrogen receptor-dependent formation of two distinct multiprotein complexes on the human pS2 gene regulatory segment. Participation of a c-fos related protein.

DNA-protein interactions around the regulatory region of the pS2 gene were studied to gain insight into the mechanisms that operate in the estrogen receptor regulated expression of this gene in the MCF-7 human breast cancer cell. Using a revised photocrosslinking technology in combination with gel retardation assays, two distinct multiprotein DNA complexes were shown to assemble in an estrogen receptor-dependent process. Immunological analysis demonstrated the participation of both the estrogen receptor and a c-fos related protein in the formation of these complexes. The results support a model of estrogen receptor function in which the receptor facilitates the formation of multiprotein complexes at DNA sites that can regulate the transcription of a hormone responsive gene by RNA polymerase II and any additional general transcription machinery. These receptor-containing complexes are referred to as "receptorsomes."

Polyclonal antibodies from rabbits and chickens against the estrogen receptor and related peptides. Use in the affinity isolation of estrogen receptors and the retrieval of chromatin fragments associating with estrogen receptors.

Polyclonal antibodies from chickens and rabbits have been prepared against polypeptides representing two regions of the human estrogen receptor (hER). The estrogen receptor (ER) peptides used as antigens were overproduced in Escherichia coli. When indicated, the antibodies were affinity purified using resins to which the antigens contained in bacterial inclusion bodies had been coupled in high yield to epoxy-activated agarose. The antibodies recognize denatured human, bovine, rat, and rabbit ER in immunoblotting experiments. Immuno-precipitation of native ER protein was readily accomplished using rabbit antisera and immobilized protein A. The chicken antibodies, available in larger quantities, were also useful for immunoisolation after coupling to agarose. With the use of these reagents, the selective retrieval of chromatin fragments from MCF-7 cells that interact with ER has been achieved.

v-erbA and citral reduce the teratogenic effects of all-trans retinoic acid and retinol, respectively, in Xenopus embryogenesis.

Treatment of late blastula/early gastrula stage Xenopus embryos with all-trans retinoic acid results in disruption of the primary body axis through effects on both mesoderm and neuroectoderm. This effect of retinoic acid, coupled with the known presence of retinoic acid in Xenopus embryos has led to the proposal that retinoic acid may be an endogenous morphogen providing positional information in early development. To further elucidate the role of retinoic acid in early Xenopus development, we have attempted to interfere with the retinoic acid signalling pathway both at the level of retinoic acid formation, by treatment with citral (3,7-dimethy-2,6-octadienal), and at the level of nuclear retinoic acid receptor function, by microinjection of v-erbA mRNA. The feasibility of this approach was demonstrated by the ability of citral treatment and v-erbA mRNA injection to reduce the teratogenic effects of exogenous retinol and retinoic acid, respectively, in early Xenopus development. Interestingly, v-erbA mRNA injection and citral treatment of gastrula stage embryos resulted in tadpoles with a similar set of developmental defects. The defects were chiefly found in tissues that received a contribution of cells from the neural crest, suggesting that at least a subset of neural crest cells may be sensitive to the endogenous level of retinoic acid. In accord with this proposal, it was found that the expression patterns of two early markers of cranial neural crest cells, Xtwi and XAP-2, were altered in embryos injected with v-erbA mRNA. These results indicate that structures in addition to the primary axis are regulated by retinoic acid signalling during early Xenopus development.

A ligand-induced conformational change in the estrogen receptor is localized in the steroid binding domain.

Upon binding estrogen, the estrogen receptor (ER) is proposed to undergo some form of conformational transition leading to increased transcription from estrogen-responsive genes. In vitro methods used to study the transition often do not separate heat-induced effects on the ER from estrogen-induced effects. The technique of affinity partitioning with PEG-palmitate was used to study the change in the hydrophobic surface properties of the ER upon binding ligand with and without in vitro heating. Upon binding estradiol (E2), the full-length rat uterine cytosolic ER undergoes a dramatic decrease in surface hydrophobicity. The binding of the anti-estrogen 4-hydroxytamoxifen (4-OHT) results in a similar decrease in surface hydrophobicity. These effects are independent of any conformational changes induced by heating the ER to 30 degrees C for 45 min. The use of the human ER steroid binding domain overproduced in Escherichia coli (ER-C) and the trypsin-generated steroid binding domain from rat uterine cytosolic ER demonstrates that the decrease in surface hydrophobicity upon binding E2 or 4-OHT is localized to the steroid binding domain. Gel filtration analysis indicates that the change in surface hydrophobicity upon binding ligand is an inherent property of the steroid binding domain and not due to a ligand-induced change in the oligomeric state of the receptor. The decrease in surface hydrophobicity of the steroid binding domain of the ER upon binding E2 or 4-OHT represents an early and possibly a necessary event in estrogen action and may be important for "tight" binding of the ER in the nucleus.

Overproduction of full-length and truncated human estrogen receptors in Escherichia coli.

The full-length human estrogen receptor (hER) as well as two overlapping peptides of hER were overproduced in Escherichia coli JM109 cells, using the inducible pIC vector system. The N-terminal receptor peptide contains the DNA-binding domain as well as the hinge region, whereas the C-terminal peptide contains the same hinge region and the hormone-binding domain. Typically, 1-6 mg of estrogen receptor (ER) peptides can be recovered from 1 L E. coli cell cultures. The majority of the overexpressed proteins are found in inclusion bodies, which allow the isolation of ER peptides in high yields and of 50-80% purity. Induction for short time periods at 10 microM inducer yielded up to 50% of the ER peptides in soluble form with full biological activity. Both the intact receptor and the C-terminal fragment specifically bound estrogens and antiestrogens, whereas ER peptides that contained the DNA-binding domain were retained on a DNA-agarose resin.