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Biomedical subjects

M J Tsai

Publications and source records attributed to M J Tsai.

At least 19 recordsLinked to original sources
PubMed1992

Ligand-dependent conformational changes in the progesterone receptor are necessary for events that follow DNA binding.

Hormones and antihormones induce related, but distinct, conformational changes in the progesterone receptor [Allan, G. F., Leng, X., Tsai, S. Y., Weigel, N. L., Edwards, D. P., Tsai, M.-J. & O'Malley, B. W. (1992) J. Biol. Chem. 267, 19513-19520]. In both cases the conformational change precedes the dissociation of heat shock proteins and binding to DNA. We have now investigated the steps in hormone action which are dependent upon this conformational change. We show that in the absence of ligand, monoclonal antibodies directed against different regions of the progesterone receptor can induce high-affinity binding to its response element in vitro. This antibody-induced DNA binding is presumably facilitated by enhanced dimerization of receptor monomers. However, antibodies do not induce the hormone-specific conformational change in the progesterone receptor and do not induce in vitro transcription by the receptor. In contrast, the antiprogestin ZK98299, which inhibits receptor binding to DNA, fully induces the antihormone-specific conformational change. Thus, our data imply that steroids induce a conformational change in their receptors which is necessary for events subsequent to DNA binding, most likely for transactivation.

Antigen-Antibody Reactions↗
PubMed1992

Kindred S thyroid hormone receptor is an active and constitutive silencer and a repressor for thyroid hormone and retinoic acid responses.

Mutations in the gene encoding the human thyroid hormone receptor beta (hTR beta) have been associated with generalized thyroid hormone resistance (GTHR). However, the molecular basis by which the receptor mutants cause the clinical symptoms is largely unknown. We show here that the beta form of the human receptor possesses, in addition to hormone-dependent activation, the ability to repress basal-level activity of a target promoter. This silencing function is localized in the carboxyl-terminal part of the receptor and can be transferred to a heterologous DNA binding domain. This mode of silencing is therefore distinct from inhibition by competition with activator proteins on DNA. We show that two receptor mutants isolated from patients with GTHR are impaired in transcriptional activation but fully retain the silencing function, which enforces dominant negative regulation by the receptor. Interestingly, the kindred S receptor (hTR delta 332) acts as a constitutive repressor with a strong silencing ability similar to that of the v-erbA oncogene product. We also provide evidence for distinct transcriptional regulatory properties of both proteins. Finally, we show that both thyroid hormone- and retinoic acid-responsive genes are potentially repressed to generate the clinical manifestations of the GTHR syndrome. Our findings suggest that silencing plays an important role in the phenotypic expression of the symptoms in patients with GTHR.

Binding Sites↗
PubMed1992

Hormone and antihormone induce distinct conformational changes which are central to steroid receptor activation.

Antihormones are potent antagonists of hormone action in vivo, but the mechanism underlying this antagonism is not understood. Several steroid hormones transform (activate) their receptors from a cytosolic, non-DNA binding 8 S sedimentation form to a nuclear, DNA binding 4 S form. Transformation is accompanied by the loss of associated heat shock proteins. We have previously demonstrated that an additional hormone-dependent step, separate from heat shock protein removal, is required for activation of the human progesterone receptor. We have devised an assay in which the human progesterone receptor translated in vitro binds to its specific response element in a hormone-dependent manner. As assessed by limited proteolytic digestion, hormone treatment of the nascent receptor induces a dramatic conformational change within the protein. The conformational change occurs in the absence of DNA and renders the entire ligand binding domain resistant to digestion by proteases. A number of antiprogestins, including RU486, induce an equally dramatic, but distinct, structural alteration of the ligand binding domain. The distinction centers upon the final 30 to 40 amino acids at the carboxyl terminus. The conformational change can be induced by ligand prior to dissociation of the 8 S complex and is not induced by heat shock protein removal in the absence of hormone. Remarkably, virtually identical hormone-induced conformational changes were detected following proteolytic analysis of in vitro translated retinoic acid receptors. Our data indicate that the sole necessary event in the activation of steroid receptors is conformational modification by the ligand. Furthermore, we conclude that transcriptional inactivation of steroid receptors by antihormones involves the induction of an inappropriate structural conformation at the extreme carboxyl terminus of the ligand binding domain.

Base Sequence↗
PubMed1992

Members of the steroid hormone receptor superfamily interact with TFIIB (S300-II).

The S300-II factor was discovered as an activator of ovalbumin gene transcription with the chicken ovalbumin upstream promoter-transcription factor (COUP-TF, Sagami, I., Tsai, S. Y., Wang, H., Tsai, M.-J., and O'Malley, B. W. (1986) Mol. Cell. Biol. 6, 4259-4267). Although S300-II does not bind DNA selectively, it stabilizes the binding of COUP-TF to its ciselement (Tsai, S. Y., Sagami, I., Wang, H., Tsai, M.-J., and O'Malley, B. W. (1987) Cell 50, 701-709). Purified S300-II is also required for steroid receptor-activated transcription. Cloning and sequencing of S300-II showed that it is the general transcription factor TFIIB. Specific protein-protein interactions between recombinant S300-II/TFIIB and three members of the steroid hormone receptor superfamily, COUP-TF, estrogen receptor, and progesterone receptor, indicate that S300-II/TFIIB is one of the targets of these transactivators. Interestingly, a truncated estrogen receptor construct containing only the N-terminal transcription activation function 1 did not interact with S300-II/TFIIB in our assay, revealing that individual transcription activation functions of a single steroid hormone receptor may contact different targets. Demonstration of a direct association of S300-II/TFIIB and COUP-TF, independent of additional "adaptor" proteins, suggests that members of the steroid hromone receptor superfamily facilitate the transcription of activated genes at least in part via protein-protein interactions with the general transcription factor TFIIB.

Amino Acid Sequence↗
PubMed1992

The mechanism of RU486 antagonism is dependent on the conformation of the carboxy-terminal tail of the human progesterone receptor.

The human progesterone receptor form B (hPR-B) was expressed in Saccharomyces cerevisiae together with a specific reporter plasmid. To understand the mechanism underlying antagonist ligand activity, libraries of hormone binding domain (HBD)-mutated hPR-B molecules were prepared. A mutant receptor was identified that had lost the ability to bind either progesterone or R5020; it could still bind RU486 and, surprisingly, fully activated transcription in the presence of this "antagonist" and other antiprogestins. When this receptor mutant was assayed in mammalian cells, RU486 again demonstrated agonistic activity. Sequence analysis indicated that the mutant phenotype was due to truncation of the carboxy (C)-terminal 42 aa. We conclude that amino acids in the extreme C-terminal region are required for the receptor to bind progesterone, while antagonists bind to a site located more N-terminal of the HBD. Our results suggest that the extreme C-terminal region of the receptor contains an inhibitory function that silences receptor transactivation in the absence of agonist and in the presence of antagonist.

Amino Acid Sequence↗
PubMed1992

Ligand and DNA-dependent phosphorylation of human progesterone receptor in vitro.

The progesterone receptor (PR), like other members of the steroid receptor family, is a ligand-induced transcription factor. We have demonstrated previously that progesterone-induced binding of PR to a progesterone response element (PRE)-linked promoter stimulates RNA synthesis from that promoter in a cell-free transcription extract. It has been established that a hormone-mediated activation of PR beyond the removal of associated heat shock proteins is essential for efficient transactivation of the target gene. We now report that treatment with hormone leads rapidly to multiple phosphorylations of both the A and B forms of human PR in a HeLa nuclear extract. The putative kinase is present in the transcriptional extract but fails to phosphorylate the receptor significantly in the absence of specific hormone or DNA. Efficient phosphorylation of the PR occurs only in the presence of PREs, indicating that ligand-induced binding of PR to its cognate DNA response element makes it a preferred substrate for the kinase. The kinetics of the phosphorylation reaction overlap the kinetics of hormone-dependent RNA synthesis from a PRE-containing target promoter in vitro. We postulate that ligand and DNA-dependent phosphorylation of PR is an important functional event in the process leading to receptor-mediated transactivation of target genes.

Cell-Free System↗
PubMed1992

Hormone activation of baculovirus expressed progesterone receptors.

Human and chicken progesterone receptors (A form) were overproduced in a baculovirus expression system. These recombinant progesterone receptors were full-length bound progesterone specifically and were recognized by monoclonal antibodies, AB52 and PR22, specific for human and chicken progesterone receptor, respectively. In gel retardation studies, binding of recombinant human and chicken progesterone receptors to their progesterone response element (PRE) was specific and was enhanced in the presence of progesterone. Binding of human progesterone receptor to the PRE was also enhanced in the presence of the antiprogestin, RU486, but very little effect was observed in the presence of estradiol, dexamethasone, testosterone, and vitamin D. In our cell-free transcription system, human progesterone receptor induced transcription in a receptor-dependent and hormone-activable manner. Receptor-stimulated transcription required the presence of the PRE in the test template and could be specifically inhibited by excess PRE oligonucleotides. Furthermore, chicken progesterone receptor also induced in vitro transcription in a hormone-activable manner. These results demonstrate that steroid receptors overexpressed in a baculovirus expression system are functional and exhibit steroid-responsive binding and transcription. These observations support our present understanding of the mechanism of steroid receptor-regulated gene expression and provide a technological format for studies of the role of hormone and antihormone in altering gene expression.

Animals↗
PubMed1992

Identification of cis- and trans-acting factors regulating the expression of the human insulin receptor gene.

The functional organization of the human insulin receptor (hIR) promoter was analyzed by deletion mutagenesis and protein-DNA interaction studies. A series of deletion mutants was expressed transiently in two human hepatocytes, HepG2 and PLC. The results revealed that the promoter region between -692 and -345 is essential for efficient transcription of the hIR gene. Multiple trans-acting factors were identified by band shift and footprinting analyses. Sp1 binds to a cluster of GC boxes and two GGGAGG hexamers locating at -637 to -594. Adjacent to GC boxes, there are two regions, from -550 to -530 and from -522 to -503, which bind to two novel factors, IRNF-I and IRNF-II. These two factors are distributed differentially in different cell lines. Linker scanning mutations on GC, GA boxes, or the IRNF-I binding site significantly decreased the transcriptional activity, indicating that IRNF-I and Sp1 are important for hIR promoter activity. In addition, we demonstrated that glucocorticoid-dependent transcriptional induction of hIR mRNA in vivo is conferred by a glucocorticoid response element in the hIR promoter. Taken together, these results imply that transcription of the human insulin receptor gene is regulated by multiple protein-DNA interactions occurring within the defined promoter region.

Animals↗
PubMed1992

Molecular pathways of steroid receptor action.

Over the past two decades, a great deal of evidence has accumulated in favor of the hypothesis that steroid hormones act at the level of nuclear DNA to regulate gene expression (Jensen EV, Suzuki T, Kawashima T, Stumpf WE, Jungblut PW, DeSombre ER, Proc Natl Acad Sci USA 1968; 59:632-638; Gorski J, Toft D, Shyamala G, Smith D, Notides A, Rec Prog Horm Res 1968; 24:45-80; O'Malley BW, Means AR, Science 1974; 183:610-620; O'Malley BW, Roop DR, Lai EC, Nordstrom JL, Catterall JF, Swaneck GE, Colbert DA, Tsai M-J, Dugaiczyk A, Woo SLC, Rec Prog Horm Res 1979; 35:1-46). The earliest studies were qualitative and involved experiments showing that steroid hormones (1) cause accumulation of new species of hybridizable RNAs that did not exist prior to stimulation; (2) cause stimulation of synthesis of new specific proteins; (3) cause a corresponding increase in the cellular levels of specific mRNAs; and (4) stimulate the rate of transcription of certain nuclear genes (O'Malley BW, McGuire WL, Kohler PO, Korenman SG, Rec Prog Horm Res 1969; 25:105-000). At that time, the early 1970s, the primary pathway for steroid hormone action was defined as follows: steroid----(steroid-receptor)----(steroid-receptor-DNA)----mRNA----fu nct ional response (O'Malley BW, Roop DR, Lai EC, Nordstrom JL, Catterall JF, Swaneck GE, Colbert DA, Tsai M-J, Dugaiczyk A, Woo SLC, Rec Prog Horm Res 1979; 35:1-46. Steroid enters cells by passive diffusion and allosterically activates receptors in either the cytoplasm or nucleus. The activated receptor binds usually at the 5'-flanking region of target genes and stimulates transcription and protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Chicken ovalbumin upstream promoter transcription factor (COUP-TF) dimers bind to different GGTCA response elements, allowing COUP-TF to repress hormonal induction of the vitamin D3, thyroid hormone, and retinoic acid receptors.

Alignment of natural chicken ovalbumin upstream promoter transcription factor (COUP-TF) response elements shows that, in addition to the predominant direct repeat of the GGTCA motif with a 2-bp spacing, there are other functional COUP elements with variations in the GGTCA orientation and spacing. We systematically analyzed the binding of in vitro-synthesized COUP-TFs and showed that COUP-TF is capable of binding to oligonucleotides containing both direct repeats and palindromes and with different spacings of the GGTCA repeats. Subsequently, we analyzed four possible mechanisms proposed to explain how COUP-TF could bind to these spatial variations of the GGTCA repeat. We demonstrated that the functional DNA-binding form of COUP-TF is a dimer which requires two GGTCA half-sites to bind DNA. We demonstrated that the COUP-TF dimer undergoes a remarkable structural adaptation to accommodate binding to these spatial variants of the GGTCA repeats. A functional consequence of the promiscuous DNA binding of COUP-TF is its ability to down-regulate hormonal induction of target gene expression by other members of the steroid-thyroid hormone receptor superfamily such as the vitamin D3, thyroid hormone, and retinoic acid receptors. Our data indicate that COUP-TF may have an important role in hormonal regulation of gene expression by these receptors.

Animals↗
PubMed1979

Effect of estrogen on ovalbumin gene expression in differentiated nontarget tissues.

By use of cloned DNA fragments as probes, low levels of ovalbumin RNA sequences (structural and intervening sequences) were detected in nuclear RNA extracts of nontarget tissues, such as liver, spleen, brain, and heart of chicks. The expression of the ovalbumin gene sequences was hormone dependent. In estrogen-stimulated chicks, a low level of ovalbumin RNA sequences, ranging from 0.2 to 0.7 molecule per cell, was present in nontarget tissues while less than 0.01 molecule per cell could be found in the same tissues of unstimulated chicks. A significant amount of the ovalbumin mRNA sequences was also found in polysomes of liver and brain. The ovalbumin mRNA sequences could be translated into proteins which were only localized in a few cells among the entire population of liver cells as determined by an immunocytochemical assay. These results suggest that there are some cells in liver, spleen, heart, and brain which can respond to hormone stimulation and produce ovalbumin mRNA and its translational product.

Animals↗
PubMed1979

Distribution of RNA transcripts from structural and intervening sequences of the ovalbumin gene.

A study was made of the function of the intervening sequences in the ovalbumin gene, Radioactively labeled DNA probes for the intervening sequences were prepared and RNA's were isolated from whole cells, nuclei, and polysomes of estrogen-stimulated chick oviducts. The concentrations of messenger RNA (mRNA) transcripts from ovalbumin structural sequences (mRNAov) and transcripts corresponding to intervening sequences were then estimated by hybridization to cloned DNA probes. Oviduct tissue contains approximately 58,000 molecules of mRNAov sequences per tubular gland cell and most of these sequences are present in the cytoplasm. In contrast, there are 200 to 300 molecules of RNA per cell which are transcribed from the intervening sequences of the natural ovalbumin gene and almost all of these are found in the nucleus. The difference in distribution of structural and intervening sequence transcripts suggests that, unlike mature mRNA, the intervening sequences are not preferentially transported to cytoplasmic polysomes.

Animals↗
PubMed1979

The ovalbumin gene: transcriptional regulation by estrogen.

De novo synthesis of RNA sequences corresponding to intervening as well as to structural sequences of the ovalbumin gene have been detected in isolated oviduct nuclei. Their presence in the nuclear transcripts and their time course of induction support the hypothesis that transcription of structural and intervening sequences of the natural ovalbumin gene are regulated by steroid hormones. These results are in agreement with out previous demonstration of high-molecular-weight species of ovalbumin RNA in nuclei that contain structural as well as intervening RNA sequences and are thus likely precursors to mature cytoplasmic mRNAov. Analysis of the size of in vivo nuclear RNA by gel electrophoresis under denaturing conditions, revealed that withdrawal of hormone depletes the level of high molecular weight ovalbumin RNA as well as that of nature mRNAov and that readministration of estrogen induces the accumulation of both species. These results are consistent also with transcriptional regulation of the ovalbumin gene. In addition, they rule out the possibility that the rapid accumulation of mature mRNAov after secondard stimulation results from processing of ovalbumin RNA precursors that might have been stored in the withdrawn oviduct. We conclude that steroid hormones exert a primary effect at the level of gene transcription. Following this event, a series of coordinated cellular responses may occur which involve RNA processing, mRNA transport to the cytoplasm, protein synthesis and mRNA degradation. The final consequence of this network of molecular reactions is the induced cellular function inherent to a specific steroid hormone.

Animals↗