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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

Modulation of progesterone receptor binding to progesterone response elements by positioned nucleosomes.

In cells, steroid hormone receptors interact with target enhancer elements on nucleosomes to regulate transcription of genes. To elucidate how nucleosomes can potentially regulate the interactions of steroid receptors with steroid response elements, we have examined the effects of nucleosome positioning and histone source on the binding of the progesterone receptor to DNA elements on nucleosomes reconstituted in vitro. We find that the affinity of the receptor for its response element is dependent on the position of the element within the nucleosome, but not on the histone source, active or inactive chromatin. Our results suggest that the strength of DNA-histone interactions within the nucleosome modulates the binding of progesterone receptor to response elements. Thus, nucleosome positioning is likely to influence the function of steroid receptors in vivo.

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↗
PubMed1992

Identification of novel steroid-response elements.

A rapid method for defining novel steroid-responsive elements has been developed. Large libraries of degenerate oligonucleotides were analyzed using a yeast-based screen to identify estrogen-responsive DNA sequences. From a library of 40,000 recombinants, seven estrogen-responsive clones were identified. When sequenced, these elements showed remarkable diversity and were different from the consensus vitellogenin A2 ERE. One surprising result was the presence of the two half sites as direct repeats in some of the clones. This implies that in vivo estrogen receptor can bind and transactivate yeast genes through response elements in which the two half sites align as direct repeats. This protocol requires no purified protein and specifically selects for functional response elements. It has a wide application in the study of any transcription factor/DNA interaction.

Base Sequence↗
PubMed1991

Studies on the mechanism of functional cooperativity between progesterone and estrogen receptors.

Steroid response elements (SREs) cooperate with many different cis-acting elements including NF-1 sites, CACCC boxes, and other SREs to induce target gene expression (Schule, R., Muller, M., Otsuka-Murakami, H., and Renkawitz, R. (1988) Nature 332, 87-90; Strahle, U., Schmid, W., and Schutz, G. (1988) EMBO J. 7, 3389-3395). Induction of gene expression can be additive or synergistic with respect to the level of activation by either transactivators. Two mechanisms have been proposed for how synergism occurs: 1) cooperative binding of transcriptional activators to DNA or 2) simultaneous interaction of individually bound activators with a common target protein. We have shown previously that cooperative binding of receptors is important for synergism between two progesterone response elements (PREs). Here we showed that an estrogen response element (ERE) and a PRE can also functionally cooperate and this synergism between an ERE and a PRE is not contributed by cooperative DNA binding. Furthermore, we have demonstrated that the activation domains of the progesterone receptor (PR) (C1Act) are required for synergism between two PREs and sufficient for confirming cooperative binding. However these two activation domains of PR are not sufficient for synergism between an ERE and a PRE. Additional regions within the NH2-terminal and COOH-terminal domains are also required for synergistic interaction between two heterologous SREs.

Base Sequence↗
PubMed1991

Cell-specific and ubiquitous factors are responsible for the enhancer activity of the rat insulin II gene.

Pancreatic beta-cell-specific expression of the insulin gene is mediated, at least in part, by an enhancer element termed the rat insulin promoter element 3 (RIPES) found within the rat insulin II gene between positions -126 and -86. Here we identify three distinct factors interacting with RIPE3, namely 3a1, 3a2, and 3b1, which bind to the sequences between -100 to -90, -108 to -99, and -115 to -107, respectively. Factors 3a1 and 3b1 are beta-cell specific whereas 3a2 is ubiquitously distributed. The 3a1 site contains the consensus binding sequence (CANNTG) for a group of DNA-binding proteins called basic-helix-loop-helix proteins. We showed in this study that the 3a1 binding activity contains E12/E47, a member of the basic-helix-loop-helix protein family, or an E12/E47-like protein. Sequence comparison of the 3a2 and 3b1 binding sites suggest that they are unique and may bind to novel transcription factors. Mutation analysis of each individual binding site in transient expression experiments indicates that all of the three binding sites contribute to the enhancer activity of the RIPE3 in beta-cells. Mutation in any one of the three binding sites not only disrupts binding of the corresponding factor but decreases RIPE3 enhancer activity by 4-7-fold. The results suggest that interactions among the 3a1, 3a2, and 3b1 factors are required for maximum enhancer activity of the RIPE3 in insulin-producing cells.

Animals↗
PubMed1991

Antiestrogen can establish nonproductive receptor complexes and alter chromatin structure at target enhancers.

We describe in this report experiments in vivo that demonstrate that antiestrogens promote DNA binding of the estrogen receptor without efficiently inducing transcription. When the receptor is modified to carry a foreign unregulated transactivation domain, transcription can be induced efficiently by both estrogen and antiestrogens. Under apparent saturation conditions, antihormone-receptor complexes binding to responsive enhancer elements elicit only a low level of transcription. In addition, we show that both estrogen and an antiestrogen, nafoxidine, effect very similar alterations in chromatin structure at a responsive promoter. These results indicate that in vivo steroid receptor action can be regulated subsequent to the DNA binding step, by regulating interactions with the target transcriptional machinery. In this regard, antihormones can function by establishing receptor-DNA complexes that are transcriptionally nonproductive.

Chromatin↗
PubMed1991

Synergism between steroid response and promoter elements during cell-free transcription.

We have analyzed quantitatively the influence of distal promoter elements on steroid-responsive gene expression in vitro. Functional synergism between enhancer and distal promoter elements was examined using two model promoters, one containing a natural promoter (mouse mammary tumor virus long terminal repeat) and one constructed artificially. Human glucocorticoid receptor (GR) expressed in baculovirus induces transcription from a mouse mammary tumor virus long terminal repeat-containing DNA template. Transcription is diminished by oligonucleotides containing a nuclear factor 1 (NF-1)-binding site or a glucocorticoid/progesterone response element. Quantitative analysis indicates that NF-1 and GR act synergistically during transcriptional activation. In contrast, efficient activation by GR or purified chick progesterone receptor of a glucocorticoid/progesterone response element-linked ovalbumin promoter does not require interaction with the chicken ovalbumin upstream promoter (COUP) element in the distal promoter. Lack of synergism is not related to enhancer strength, since the glucocorticoid/progesterone response elements can be moved further from the promoter or reduced to a single copy response element without increasing the dependence upon COUP. Strong synergism is restored following substitution of an NF-1 distal promoter element for the COUP element in this construct. Our results suggest that synergism between steroid response and distal promoter elements is dependent upon the identity of the promoter element rather than upon the inherent strength of the enhancer element.

Animals↗
PubMed1991

Steroid hormone receptors and in vitro transcription.

Steroid hormone receptors are ligand-inducible transcription factors that exhibit potent effect on gene expression in living cells. Precise dissection of their mode of action at the molecular level can best be carried out in functional cell-free systems. This article will describe the benefits of such systems and review their development up to the recent establishment of steroid receptor-dependent in vitro transcription. Subsequent advances in our knowledge of receptor function arising from the exploitation of this powerful experimental tool will be described. Particular emphasis will be placed upon two key problems: the role of steroid hormone in receptor action and the mechanisms by which steroid receptors activate gene transcription.

Animals↗