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Biomedical subjects

B M Forman

Publications and source records attributed to B M Forman.

At least 37 records · Page 2Linked to original sources
PubMed1994

Differential expression and activation of a family of murine peroxisome proliferator-activated receptors.

To gain insight into the function of peroxisome proliferator-activated receptor (PPAR) isoforms in mammals, we have cloned and characterized two PPAR alpha-related cDNAs (designated PPAR gamma and -delta, respectively) from mouse. The three PPAR isoforms display widely divergent patterns of expression during embryogenesis and in the adult. Surprisingly, PPAR gamma and -delta are not activated by pirinixic acid (Wy 14,643), a potent peroxisome proliferator and activator of PPAR alpha. However, PPAR gamma and -delta are activated by the structurally distinct peroxisome proliferator LY-171883 and linoleic acid, respectively, indicating that each of the isoforms can act as a regulated activator of transcription. These data suggest that tissue-specific responsiveness to peroxisome proliferators, including certain fatty acids, is in part a consequence of differential expression of multiple, pharmacologically distinct PPAR isoforms.

Acetophenones↗
PubMed1994

Cross-talk among ROR alpha 1 and the Rev-erb family of orphan nuclear receptors.

We have cloned Rev-erb beta, a novel isoform of the Rev-erb alpha orphan nuclear receptor. The DNA binding domains of Rev-erb alpha and beta are highly related to each other and to the retinoic acid related orphan receptor (ROR)/RZR subfamily of nuclear receptors. Indeed, we find that all three receptors bind as monomers to the sequence AATGT-AGGTCA. Whereas ROR alpha 1 constitutively activates transcription through this sequence, both isoforms of Rev-erb are inactive. When coexpressed, both Rev-erb isoforms suppress the transcriptional activity of ROR alpha 1. Our data define Rev-erb and ROR/RZR as a family of related receptors with opposing activities on overlapping regulatory networks.

Amino Acid Sequence↗
PubMed1993

Functional ecdysone receptor is the product of EcR and Ultraspiracle genes.

Although the biological activity of the insect moulting hormone ecdysone, is manifested through a hormonally regulated transcriptional cascade associated with chromosomal puffing, a direct association of the receptor with the puff has yet to be established. The cloned ecdysone receptor (EcR) is by itself incapable of high-affinity DNA binding or transcriptional activation. Rather, these activities are dependent on heterodimer formation with Ultraspiracle (USP) the insect homologue of vertebrate retinoid X receptor. Here we report that native EcR and USP are co-localized on ecdysone-responsive loci of polytene chromosomes. Moreover, we show that natural ecdysones selectively promote physical association between EcR and USP, and conversely, that high-affinity hormone binding requires both EcR and USP. Replacement of USP with retinoid X receptor produces heterodimers with distinct pharmacological and functional properties. These results redefine the ecdysone receptor as a dynamic complex whose activity may be altered by combinatorial interactions among subunits and ligand.

Animals↗
PubMed1993

Conformational changes in chicken thyroid hormone receptor alpha 1 induced by binding to ligand or to DNA.

A classic model of steroid/thyroid hormone receptor activation postulates that a conformational change or "transformation" occurs upon ligand binding as a first step toward regulation of gene transcription. In order to test this model, physical studies have been carried out using purified full-length chicken thyroid hormone receptor alpha 1 (cT3R-alpha 1) expressed in Escherichia coli. Circular dichroism spectroscopic studies reveal that cT3R-alpha 1 adopts a different conformation upon specific binding to a cognate ligand triiodothyroacetic acid as well as to a thyroid hormone response element, an idealized inverted repeat AGGTCA TGACCT. These results suggest that cT3R-alpha 1 may adopt distinct conformations whether free or bound to ligand or to DNA. These states may reflect the changes in the conformation of steroid/thyroid hormone receptors in the signal transduction pathway.

Animals↗
PubMed1992

Half-site spacing and orientation determines whether thyroid hormone and retinoic acid receptors and related factors bind to DNA response elements as monomers, homodimers, or heterodimers.

The receptors for thyroid hormone (T3R) and retinoic acid (RAR) are members of a nuclear receptor subfamily that are capable of recognizing similar DNA sequences. Native response elements for T3R and RAR consist of two or more putative half-site binding motifs organized as imperfect direct or inverted repeats separated by different sized nucleotide gaps. To clarify how T3R, RAR, and related factors recognize DNA response elements, we analyzed the interaction of purified receptors with a series of inverted and direct repeats of an idealized AGGTCA half-site separated by different sized nucleotide gaps. Our results indicate that RAR and T3R can bind to half-sites as monomers and, depending on the orientation and distance between half-sites, also bind as homodimers or T3R-RAR heterodimers. T3R also binds to certain DNA elements as a heterodimer with one or more nuclear factors from eucaryotic cells. Thus, the orientation and spacing of half-sites play a central role in determining which configuration of receptors and nuclear factors will interact with a specific DNA element. This along with the ability of these factors to participate in reversible protein-protein interactions serve to broaden and diversify the responses mediated by T3R, RAR, and related members of this nuclear receptor subfamily.

Base Sequence↗
PubMed1992

Capacity for cooperative binding of thyroid hormone (T3) receptor dimers defines wild type T3 response elements.

Thyroid hormone response elements (T3REs) have been identified in a variety of promoters including those directing expression of rat GH (rGH), alpha-myosin heavy chain (rMHC), and malic enzyme (rME). A detailed biochemical and genetic analysis of the rGH element has shown that it consists of three hexamers related to the consensus [(A/G)GGT(C/A)A]. We have extended this analysis to the rMHC and rME elements. Binding of highly purified thyroid hormone receptor (T3R) to T3REs was determined using the gel shift assay, and thyroid hormone (T3) induction was measured in transient tranfections. We show that the wild type version of each of the three elements binds T3R dimers cooperatively. Mutational analysis of the rMHC and rME elements identified domains important for binding T3R dimers and allowed a direct determination of the relationship between T3R binding and function. In each element two hexamers are required for dimer binding, and mutations that interfere with dimer formation significantly reduce T3 induction. Similar to the rGH element, the rMHC T3RE contains three hexameric domains arranged as a direct repeat followed by an inverted copy, although the third domain is weaker than in rGH. All three are required for full function and T3R binding. The rME T3RE is a two-hexamer direct repeat T3RE, which also binds T3R monomer and dimer. Across a series of mutant elements, there was a strong correlation between dimer binding in vitro and function in vivo for rMHC (r = 0.99, P less than 0.01) and rME (r = 0.67, P less than 0.05) T3REs. Our results demonstrate a similar pattern of T3R dimer binding to a diverse array of hexameric sequences and arrangements in three wild type T3REs. Addition of nuclear protein enhanced T3R binding but did not alter the specificity of binding to wild type or mutant elements. Binding of purified T3R to T3REs was highly correlated with function, both with and without the addition of nuclear protein. T3R dimer formation is the common feature which defines the capacity of these elements to confer T3 induction.

Animals↗
PubMed1991

Oligomeric binding of T3 receptor is required for maximal T3 response.

Receptors in the thyroid-steroid hormone superfamily bind preferentially as dimers to palindromic response elements containing two hexameric half-sites. The 23-base pair rat growth hormone (rGH) T3 response element (T3RE), however, contains three hexameric binding domains, all of which are required for maximal T3 response. We examined the binding of purified T3 receptor alpha (T3R alpha), overexpressed in Escherichia coli, to wild-type and up and down mutations of the rGH T3RE to evaluate whether transcriptional potency correlates with changes in T3R binding. T3R binds to the rGH T3RE as a monomer, dimer, or higher order oligomer. Cooperative T3R dimer binding was demonstrated to two hexameric domains of the rGH T3RE arranged as either direct or inverted repeats. Decreased binding was seen with point mutations in each domain as well as with mutations which altered hexamer orientation and spacing within the site. These results demonstrate that all three hexamers of the rGH T3RE are involved in binding T3R. Occupancy of all three hexamers by T3R in the gel shift assay was observed with functional up mutations of the T3RE, increasing receptor concentration or addition of nuclear extract. The transcriptional response potencies of T3RE up or down mutants in a transient transfection assay correlated closely with T3R binding. These results confirm our earlier hypothesis that all three hexamers of the rGH T3RE bind T3R in a novel binding arrangement and provide a model for the interaction of T3R and other nuclear proteins with the DNA sequences of thyroid hormone-regulated genes.

Animals↗
PubMed1991

pEXPRESS: a family of expression vectors containing a single transcription unit active in prokaryotes, eukaryotes and in vitro.

We have constructed a family of expression vectors containing a single transcription unit that is active in Escherichia coli, eukaryotic cells, and in coupled in vitro transcription-translation systems. These vectors use the Rous sarcoma virus-long terminal repeat (RSV-LTR) as the promoter/enhancer for eukaryotic cells. In vitro transcription is made possible by inclusion of a bacteriophage T7 promoter. This same promoter is actively transcribed in E. coli that produce T7 RNA polymerase. Other features of this transcription unit include a high-efficiency eukaryotic translation start codon, a phage f1 origin of DNA replication for site-directed mutagenesis and a three-frame stop codon that facilitates C-terminal deletion mutagenesis. We term this vector family, pEXPRESS.

Avian Sarcoma Viruses↗
PubMed1991

A retinoic acid response element is part of a pleiotropic domain in the phosphoenolpyruvate carboxykinase gene.

Several hormones, including insulin, glucagon, and glucocorticoids, regulate the expression of the rate-limiting gluconeogenic enzyme, phosphoenolpyruvate carboxykinase [GTP: oxaloacetate carboxy-lyase (transphosphorylating); EC 4.1.1.32; PEPCK] in liver. In this report we demonstrate that retinoic acid (RA) also regulates PEPCK expression by inducing a 3-fold increase in the rate of transcription of the PEPCK gene. A RA response element located between -468 and -431 in the PEPCK promoter mediates a 7-fold increase in expression of a chimeric construct containing the basal PEPCK promoter ligated to the chloramphenicol acetyltransferase reporter gene. This element confers RA responsiveness through the heterologous thymidine kinase promoter and functions relatively independent of position and orientation. An 18-base-pair core sequence (-451 to -434) (i) mediates an effect of RA on PEPCK gene expression and contains motifs found in two other RA response elements; (ii) corresponds to AF1, an accessory factor element that is an integral component of the complex glucocorticoid response unit in the PEPCK gene promoter; (iii) is in a region involved in the developmental expression of the PEPCK gene; and (iv) shows homology to elements involved in the tissue-specific regulation of genes, including the hepatic apolipoprotein genes and the alpha 1-antitrypsin gene.

Animals↗
PubMed1991

Specificity of a retinoic acid response element in the phosphoenolpyruvate carboxykinase gene promoter: consequences of both retinoic acid and thyroid hormone receptor binding.

The ability of a retinoic acid (RA) response element (RARE) in the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter to mediate effects of either RA or thyroid hormone (T3) on gene expression was studied. Fusion gene constructs consisting of PEPCK promoter sequences ligated to the chloramphenicol acetyltransferase (CAT) reporter gene were used for this analysis. While T3 induced CAT expression to a small degree (about twofold) when such constructs were transiently transfected into H4IIE rat hepatoma cells, along with an expression vector encoding the alpha subtype of the T3 receptor (TR), this effect was mediated by promoter sequences distinct from the PEPCK RARE. Although TRs were capable of binding the PEPCK RARE in the form of putative monomers, dimers, and heterodimers with RA receptors (RARs), this element failed to mediate any positive effect of T3 on gene expression. In contrast, the PEPCK RARE mediated six- to eightfold induction of CAT expression by RA. When TRs were coexpressed along with RARs in transfected H4IIE cells, this RA induction was substantially blunted in a T3-independent manner. This inhibitory effect may be due to the binding of nonfunctional TRs or TR-RAR heterodimers to the PEPCK RARE. A model is proposed to explain the previously observed in vivo effects of T3 on PEPCK gene expression.

Animals↗
PubMed1991

Effects of varying the position of thyroid hormone response elements within the rat growth hormone promoter: implications for positive and negative regulation by 3,5,3'-triiodothyronine.

The thyroid hormone response element (T3RE) of the rat GH (rGH) promoter is located at -188 to -165 relative to the mRNA start site (TSS). Similar sites have been identified in other genes regulated by T3. We have investigated some of these T3REs in positions within the rGH promoter to assess the relative influences of DNA-binding site and position on positive and negative regulation by T3. Synthetic oligonucleotides were used with sequences from the rGH T3RE and proposed negative T3REs (nT3RE) from the rat and human alpha-subunit and rat beta TSH genes. The nT3REs were placed in the background of the wild-type rGH promoter in two positions, at -55 and down-stream of the TSS, with up- and down-mutations of the rGH T3RE. Rat GH T3RE elements were placed 700 basepairs up-stream of a basal rGH promoter and some also at the -55 and TSS positions. Constructions were tested in a transient transfection assay in rat pituitary tumor cells. Two copies of the rGHPAL (palindromic T3RE) placed 700 basepairs up-stream of the rGH promoter conferred 10-fold T3 induction. In the -55 position, the rGHPAL increased T3 induction compared to that in controls, whereas a fragment from the rat and human alpha-subunit gene in the same position reduced induction. Negative T3REs from rat beta TSH and human alpha-subunit reduced T3 induction 50% when placed at the TSS position of a rGH promoter containing an up-mutant T3RE. The T3REPAL placed at the same site increased T3 induction.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Interactions among a subfamily of nuclear hormone receptors: the regulatory zipper model.

The nuclear hormone receptors comprise a superfamily of ligand-modulated transcription factors that regulate homeostasis, reproduction, development, and differentiation. Three amino acids within the zinc finger DNA binding motif determine target gene specificity. Groups of receptors exist with similar DNA binding specificity. A complex carboxy terminal region mediates ligand binding, dimerization, and hormone-relieved transcriptional inactivation. We summarize the current understanding of these phenomena and suggest a novel model that structurally and functionally links these events. This "regulatory zipper model" may explain the mechanism by which ligand activates nuclear hormone receptors.

Amino Acid Sequence↗
PubMed1990

Dimerization among nuclear hormone receptors.

The nuclear hormone receptors comprise a superfamily of ligand-modulated transcription factors that regulate homeostasis, reproduction, development, and differentiation. The DNA-binding domain of the nuclear hormone receptors contains two zinc finger motifs and binds to response elements composed of two-half-sites separated by a variably sized gap. DNA binding specificity is accomplished by a combination of mechanisms. First, discrimination among half-site sequences is mediated by three amino acids within the first zinc finger. Second, response elements with different half-site spacing can be discriminated by five amino acids in the second zinc finger, which may act as a dimerization interface. A second dimerization signal is embedded within the ligand-binding domain of several receptors. Ligand binds to sequences adjacent to this region and enhances dimerization. It is possible that dimerization of these receptors could account for certain physiologic and pathologic conditions observed in vivo.

Animals↗
PubMed1989

Characterization of the domain structure of chick c-erbA by deletion mutation: in vitro translation and cell transfection studies.

Chicken c-erbA (Ck-c-erbA) cDNA (1250 base pairs), a cellular homologue of the avian erythroblastosis virus v-erbA gene, encodes a 408 amino acid protein which binds L-T3 and its analogs with affinities similar to that of endogenous thyroid hormone nuclear receptors. By analogy with steroid receptors, Ck-c-erbA(Met1-Val408) contains an A and B domain (amino acids 1-50); a putative DNA binding C domain (amino acids 51-118); a hydrophilic D domain (amino acids 119-189); and a putative ligand binding E domain (amino acids 187-408). To further characterize the ligand binding region of Ck-c-erbA, two deletion mutations were constructed: Ck-c-erbA(Met120-Val408) which encodes a 289 amino acid protein lacking regions A, B, and C; and Ck-c-erbA-(Met199-Val408) which encodes a 210 amino acid protein lacking regions A, B, C, D, and the first 12 amino acids of the E region. The in vitro translation products ([35S]methionine) of cDNA transcripts of a human placental c-erbA, Ck-c-erbA (Met1-Val408), and Ck-c-erbA(Met120-Val408) efficiently bind L-[125I]T3, whereas Ck-c-erbA(Met199-Val408) does not bind L-[125I]T3. In frame substitution of the last 14 C-terminal amino acids of Ck-c-erbA(Met1-Val408) for the last 7 C-terminal amino acids of v-erbA reduces but does not eliminate L[125I]T3 binding. These results indicate that a broad region of the E domain is important for ligand binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1989

A domain containing leucine-zipper-like motifs mediate novel in vivo interactions between the thyroid hormone and retinoic acid receptors.

The thyroid hormones and retinoic acid are potent modulators of differentiation, development, and gene expression. The transcriptional activities of these ligands are mediated by closely related nuclear receptors which bind and activate identical hormone responsive DNA elements. We noticed that a region within the ligand binding or E domain is well conserved between receptors for these hormones. This region contains hydrophobic heptad repeats that are structurally similar to the leucine-zipper dimerization domain. To study the function of this conserved domain, we examined the transcriptional responses of thyroid hormone receptor/c-erbA deletion mutants which lacked the heptad repeats. We previously reported that the chick c-erbA-alpha possesses hormone-independent (constitutive) activity in cells which express endogenous rat thyroid hormone receptor. We now demonstrate that this activity is abolished upon deletion of the conserved heptad repeats. This suggests that the heptad repeats mediate in vivo interactions between chick c-erbA and rat thyroid hormone receptors. To further test this hypothesis deletion mutants of chick c-erbA were constructed which contained all eight heptad repeats but which lacked the zinc-finger DNA binding domain. Although these mutants are transcriptionally inactive, they act in a dominant-negative fashion to block trans-activation by both the chick c-erbA-alpha and the endogenous thyroid hormone and retinoic acid receptors. We suggest that the heptad repeats mediate the formation of inactive mutant/wild-type hetero-dimers. Dimer formation suggests a mechanism to account for the dominant-negative phenotypes displayed by nonhormone binding variants of c-erbA, the proto-oncoprotein v-erbA and patients with the generalized thyroid hormone resistance syndrome.

Amino Acid Sequence↗
PubMed1988

Rat growth hormone gene expression. Both cell-specific and thyroid hormone response elements are required for thyroid hormone regulation.

The elements involved in mediating cell-specific and thyroid hormone stimulation of rat growth hormone gene expression have been defined by transfection studies and by nuclease footprinting. 5'-Flanking DNA extending to -104 can mediate cell-specific expression, and this is enhanced 3- to 4-fold with DNA extending to -145. Cell-specific factors, found only in rat growth hormone producing cells, bind within the -137/-107 and -95/-65 regions, and competition studies suggest that the same factor binds to both sites. The sequence A (A or T) TAAAT is found at the center of both footprints at -80 and -122, suggesting that it is a core component of the recognition sequence of the cell-specific factor. Disruption of the spatial and/or distance relationships between the two regions eliminates the enhanced level of cell-specific expression, suggesting a cooperative interaction of the proteins which bind to these elements. Sequences located between -208 and -178 can confer thyroid hormone-regulated expression when linked in either orientation in close proximity to one or both cell-specific elements. The thyroid hormone and cell-specific elements function as an enhancer-like unit and are both required to confer regulated expression to heterologous promoters. We propose that thyroid hormone acts via its receptor to enhance the function of the cell-specific element by forming a more "active" transcription complex which stimulates the level of gene expression.

Animals↗