Biomedical subjects
Publications and source records attributed to H H Samuels.
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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)
In the epidermis, retinoids regulate the expression of keratins, the intermediate filament proteins of epithelial cells. We have cloned the 5' regulatory regions of four human epidermal keratin genes, K#5, K#6, K#10, and K#14, and engineered constructs in which these regions drive the expression of the CAT reporter gene. By co-transfecting the constructs into epithelial cells along with the vectors expressing nuclear receptors for retinoic acid (RA) and thyroid hormone, we have demonstrated that the receptors can suppress the promoters of keratin genes. The suppression is ligand dependent; it is evident both in established cell lines and in primary cultures of epithelial cells. The three RA receptors have similar effects on keratin gene transcription. Our data indicate that the nuclear receptors for RA and thyroid hormone regulate keratin synthesis by binding to negative recognition elements in the upstream DNA sequences of the keratin genes. RA thus has a twofold effect on epidermal keratin expression: qualitatively, it regulates the regulators that effect the switch from basal cell-specific keratins to differentiation-specific ones; and quantitatively, it determines the level of keratin synthesis within the cell by direct interaction of its receptors with the keratin gene promoters.
Studies were conducted to determine whether the trans-acting protein Pit-1/GHF-1 can bind to and activate promoter elements in both the GH and PRL genes that are necessary for cell-specific expression. Four pituitary cell lines that differentially express the endogenous GH and PRL genes were examined for their ability to activate GH and PRL promoter constructs containing sequences necessary for cell-specific expression (CSEs). Plasmids containing one CSE, -96 PRL and -104 GH, were similarly expressed in each of the four cell lines. Of the plasmids containing two CSEs, -173 PRL was always activated to a greater extent than -145 GH, with this relative activation being stronger in GC and GH1 cells than in 235-1 and GH4C1 cells. Protein-DNA binding assays were used to show that the GH and PRL CSEs specifically bound two highly abundant nuclear proteins (31 and 33 kDa). The two proteins were present at similar levels in all four pituitary cell lines and were recognized by a Pit-1/GHF-1 antibody. In contrast, HeLa and Rat2 cells did not activate transfected GH or PRL plasmids and did not contain nuclear proteins that specifically bound to the GH and PRL CSEs. However, cotransfection of these cells with the expression vector RSV-Pit-1/GHF-1 resulted in the activation of -173 PRL and -145 GH (PRL greater than GH). HeLa cells transfected with RSV-Pit-1/GHF-1 also contained 31- and 33-kDa nuclear proteins that bound to the GH and PRL CSEs. These results show that Pit-1/GHF-1 is present at levels in pituitary cell lines that are sufficient to activate the minimal elements in both the GH and PRL promoters necessary for cell-specific expression of these genes.
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.
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.
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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)
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.
The glucocorticoid antagonist 17 alpha-methyltestosterone inhibits binding of the agonist [3H]triamcinolone acetonide ot the glocucorticoid receptor in cytosol prepared from rat pituitary tumor GH1 cells. Competitive binding studies indicate that the dissociation constant for 17 alpha-methyltestosterone is about 1 microM. After incubation of intact GH1 cells with 10 nM [3H]triamcinolone acetonide at 37 C and subsequent cell fractionation at 4 C, three glucocorticoid receptor forms are observed: cytosolic 10 S receptor, cytosolic 4 S receptor, and nuclear receptor. Concurrent incubation with 17 alpha-methyltestosterone reduces the amount of [3H]triamcinolone acetonide bound to each of these receptor forms. Ligand-exchange assays performed at 0 C in intact cells using [3H]triamcinolone acetonide show that the exchangeable antagonist is associated predominantly with cytosolic 10 S receptor. Immunochemical analysis using monoclonal antibody BuGR2 indicates that 17 alpha-methyltestosterone does not cause substantial accumulation of glucocorticoid receptors in GH1 cell nuclei and, when present together with agonist, reduces nuclear accumulation of receptor seen with agonist alone. Results from dense amino acid labeling studies show that unlike [3H]triamcinolone acetonide, 17 alpha-methyltestosterone does not reduce the total amount of cellular glucocorticoid receptor and does not reduce receptor half-life. These results are consistent with a model for glucocorticoid receptor transformation in which binding of agonist promotes the dissociation of an oligomeric 10 S cytosolic receptor protein to its DNA-binding 4 S subunit. The antagonist 17 alpha-methyltestosterone competes with agonist for binding to the 10 S cytosolic receptor but does not appear to promote dissociation of the oligomer, thus inhibiting agonist-mediated nuclear actions of the glucocorticoid receptor.
Rat GH gene expression is known to be stimulated by several factors, including thyroid hormone and GRF. This effect of GRF appears to be mediated by cAMP resulting from activation of adenylate cyclase by the peptide. The elements of the rat GH gene important for thyroid hormone stimulation and cell-specific expression have been previously mapped using gene transfection techniques. Cell-specific expression of the gene is mediated by two cell-specific elements located from -137 to -107 and from -95 to -65. Sequences mediating thyroid hormone stimulation are thought to be located between -208 and -160. In this study, using three different methods to elevate cAMP levels in cells [forskolin, a direct activator of the adenylate cyclase catalytic subunit; 8-(4-chlorophenylthio)cAMP, a nonmetabolizable cAMP analog; and isobutylmethylxanthine, a phosphodiesterase inhibitor], we show that 5'-flanking DNA of the rat GH gene can mediate stimulation by cAMP (10- to 20-fold). The cAMP-responsive region was mapped to sequences between -104 and +11, which contains the proximal cell-specific element (-95/-65) important for cell-specific expression. Either the -97/-65 or the -104/-47 region of the gene, cloned upstream of a heterologous promoter, conferred only minimal or no activation by cAMP. This suggests that these sequences are not the direct target of cAMP action or that they are insufficient alone to mediate the cAMP response. The cAMP regulatory element (TGACGTCA) is not found between - 104 and +11, and cAMP activation does not appear to act via putative AP-2 elements, since phorbol esters did not stimulate expression.(ABSTRACT TRUNCATED AT 250 WORDS)
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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.
The regulation of growth hormone gene expression by thyroid hormone in cultured GH1 cells is mediated by a chromatin-associated receptor. We have previously described a photoaffinity label derivative of 3,5,3'-triiodo-L-thyronine (L-T3) in which the alanine side chain was modified to form N-2-diazo-3,3,3-trifluoropropionyl-L-T3 (L-[125I]T3-PAL). On exposure to 254 nm UV light, L-[125I]T3-PAL generates a carbene which covalently modifies two thyroid hormone receptor forms in intact GH1 cells; an abundant 47,000 Mr species and a less abundant 57,000 Mr form. We have now synthesized similar photoaffinity label derivatives of 3,5,3',5'-tetraiodo-L-thyronine (L-T4) and 3,3',5'-triiodo-L-thyronine (L-rT3). Both compounds identify the same receptor forms in intact cells and in nuclear extracts in vitro as L-[125I]T3-PAL. Labeling by L-[125I]rT3-PAL was low and consistent with the very low occupancy of receptor by L-rT3. Underivatized L-[125I]T3 and L-[125I]T4 labeled the same receptor forms at 254 nm but at a markedly lower efficiency than their PAL derivatives. In contrast, N-bromoacetyl-L-[125I]T3, a chemical affinity labeling agent, did not derivatize either receptor form in vitro. The relative efficiency of coupling to receptor at 254 nm was L-[125I]T4-PAL greater than L-[125I]T3-PAL greater than L-[125I]T4 greater than L-[125I]T3. Although L-[125I]T4-PAL has a lower affinity for receptor than L-[125I]T3-PAL, its coupling efficiency was 5-10-fold higher. This suggests that the alanine side chain of L-[125I]T4-PAL is positioned in the ligand binding region near a residue which is efficiently modified by photoactivation. With L-[125I]T4-PAL we were able to identify three different molecular weight receptor species in human fibroblast nuclei.
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Regulation of gene expression by the thyroid hormones is thought to be mediated by a nuclear-associated receptor found in a wide variety of cells and tissues. Cellular homologues of the avian erythroblastosis virus oncogene, v-erbA, encode proteins which bind thyroid hormone with similar affinities as thyroid hormone receptors. However, it has not been shown that any of the c-erbA proteins can function as receptor and modulate thyroid hormone responsive genes. In this study, using transient expression of chimeric reporter constructs, we document that the chick fibroblast c-erbA-alpha and the human placental c-erbA-beta modulate cis-acting regulatory sequences of two thyroid hormone responsive genes; rat GH and PRL. From these results we conclude: 1) in a receptor deficient cell line (235-1) both c-erbA subtypes act as hormone-dependent modulators of PRL gene expression and hence function as thyroid hormone receptors, 2) in two different receptor containing cell lines (GH4C1 and GH1), both c-erbA proteins act in a hormone-independent fashion to regulate PRL and GH expression. This suggests that events other than ligand binding can result in formation of a c-erbA protein that modulates transcription of thyroid hormone responsive genes, 3) no qualitative functional differences were detected between alpha- and beta-c-erbA subtypes, and 4) depending on the cell-type, L-T3 acts through its endogenous receptor to stimulate (GH4C1) or suppress (GH1) expression of a chimeric PRL construct. In these cells, c-erbA expression results in the same positive or negative response as the endogenous receptor except that the response occurs in the absence of hormone. These results suggest that the endogenous receptor and the c-erbAs act by augmenting the effect of transcription factors which can positively or negatively control gene expression.
The elements that we have identified in the 5'-flanking region of the rat growth hormone gene are shown in Figure 7, and the following conclusions are drawn: 1) Cell-specific expression of the rat growth hormone gene is mediated by two CSEs, which are located from -95 to -65 and from -137 to -107.2) These CSEs bind a common cell-specific trans-acting factor (GH-CSF), which is found in growth hormone-producing cells. 3) Enhanced levels of cell-specific expression may involve a protein-protein interaction when this factor binds on the same side of the DNA helix as the two CSEs. 4) A TRE is located between -208 and -178. 5) Activation of the gene by thyroid hormone appears to require both the TRE and one of the CSEs, and both are required to confer L-T3 stimulation to several heterologous promoters. 6) Our studies support the notion that stimulation by L-T3 involves the binding of the L-T3-receptor complex to the TRE, which enhances the function of the CSEs, resulting in stimulation of growth hormone gene expression.