Glycoprotein Hormone Alpha-subunit Gene Research Articles

3,5,3′-Triiodothyronine Receptor Auxiliary Protein (TRAP) Enhances Receptor Binding by Interactions within the Thyroid Hormone Response Element

We have previously demonstrated that binding of in vitro synthesized thyroid hormone receptor (TR) to thyroid hormone response elements (TREs) is enhanced by the addition of nuclear extracts from several different cell types, suggesting that binding of TR is partially dependent on a T3 receptor auxiliary protein (TRAP). We have used the avidin-biotin complex DNA-binding assay to discriminate between regions of TREs that bind TR alone and sites that are influenced by interactions with TRAP. Mutations in the TREs from rat GH and glycoprotein hormone alpha-subunit genes show that a specific DNA sequence is required for TRAP-mediated enhancement of TR binding. Mutations in the B half-site of the rat GH TRE or in similar sequences [(T/A)GGGA] in the alpha-subunit TRE ablate the enhancement of TR binding by TRAP. Furthermore, binding of TR to a natural half-site in the TSH beta-subunit gene (bases -16 to 6), which lacks an additional AGGGA-like sequence, is not enhanced by the addition of TRAP. Binding of TR to TREs was also tested at physiological salt concentrations in the avidin-biotin complex DNA-binding assay. Binding of human TR beta to TREs decreases dramatically at 140 mM KCl compared to binding at 50 mM KCl; however, the addition of TRAP enhances the binding to almost 4-fold of basal binding, suggesting that TRAP may be important for stabilization of TR binding to TREs in the cell.

A cis-acting element located between the cAMP response elements and CCAAT box augments cell-specific expression of the glycoprotein hormone alpha subunit gene.

Several cis-acting elements interact through their cognate trans-acting factors to confer placenta-specific expression to the alpha subunit gene of glycoprotein hormones. These elements include two tandemly arranged cAMP response elements (CREs, located between base pairs (bp) -146 and -111 relative to the transcription start site), an upstream regulatory element (URE, located between -180 and -150), and a CCAAT box (located between bp -100 and -80). Here, we identify a new regulatory region, junctional regulatory element (JRE), with critical nucleotides located between the CREs and CCAAT box (bp -120 to -100). Although the binding site of the JRE abuts the 3' CRE, methylation interference assays indicate that no overlap occurs between the contact points of the proteins that bind to the JRE and CRE. This new regulatory element is highly conserved across species and contains a palindromic binding site for a 50-kDa nuclear factor(s) which is distinct from the factors that bind the URE, CRE, and CCAAT box of the alpha subunit gene. Finally, gene transfer studies suggest that, although JRE binding activity is found in several cell types, this element acts relatively cell-specifically. We conclude that this element and its cognate trans-acting factor act in conjunction with the complexes formed over the URE, CREs, and CCAAT box to enhance the placenta-specific activity of the alpha subunit promoter.

A cAMP response element in the beta 2-adrenergic receptor gene confers transcriptional autoregulation by cAMP.

The rate of transcription of the beta 2-adrenergic receptor gene is increased in response to beta-adrenergic agonist stimulation of the receptor at the cell surface. This effect is mediated by stimulation of adenylyl cyclase and elevation of intracellular cAMP levels. We have previously shown that this responsiveness to cAMP resides in the 5'-flanking region of the human beta 2-adrenergic receptor gene (Collins, S., Bouvier, M., Bolanowski, M. A., Caron, M. G., and Lefkowitz, R. J. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4853-4857). A 34-base pair sequence derived from the beta 2-adrenergic receptor promoter region (-70 to -37 base pairs), containing the sequence GTACGTCA, confers responsiveness to cAMP when present in either orientation 5' to the thymidine kinase promoter on the chloramphenicol acetyltransferase reporter gene. Overexpression of the catalytic subunit of protein kinase A fully substituted for forskolin in inducing expression through this sequence, indicating that the cAMP induction is mediated through this kinase. Mutations within the GTACGTCA sequence completely abolished the stimulation. A 43-kDa transcription factor (cAMP response element-binding protein) confers cAMP responsiveness through binding to specific sequences. In gel mobility shift assays, purified cAMP response element-binding protein bound to the 34-base pair oligonucleotide from the beta 2-adrenergic receptor gene with an affinity similar to that for the well-characterized cAMP response element from the human glycoprotein hormone alpha-subunit gene, and failed to bind to mutated elements. Thus, positive autoregulation of the beta 2-adrenergic receptor gene appears to occur through receptor-mediated stimulation of adenylyl cyclase, with consequent activation of cAMP response element-binding protein and stimulation of beta 2-adrenergic receptor gene transcription. These results demonstrate a novel mechanism by which a receptor (beta 2-adrenergic receptor) stimulatory for adenylyl cyclase can exert positive feedback regulation on its own expression.

Different Combinations of Regulatory Elements May Account For Expression of the Glycoprotein Hormone α-Subunit Gene in Primate and Horse Placenta

Expression of the glycoprotein hormone alpha-subunit gene occurs in the pituitaries of all mammals and in the placentas of primates and horses. In humans, tandem cAMP response elements (CREs), located in the proximal promoter-regulatory region of the alpha-subunit gene, act together with an adjacent upstream regulatory element to confer placenta-specific expression. Here, we report that the alpha-subunit genes of Old World Monkeys contain a single functional CRE. This suggests that tandem CREs are unique to higher primates and humans and are not absolutely required for placenta-specific expression. In contrast, the comparable promoter-regulatory region of the horse alpha-subunit gene lacks a functional CRE but appears to retain a functional upstream regulatory element. This suggests that acquisition of placenta-specific expression of the alpha-subunit gene occurred independently in these distantly related mammals. As a result, different combinations of cis-acting elements may explain why expression of the alpha-subunit gene only occurs in placenta of primates and horses.

Induction by cycloheximide of the glycoprotein hormone alpha-subunit gene in human tumor cell lines and identification of a possible negative regulatory factor.

The steady-state level of mRNA encoding the glycoprotein hormone alpha-subunit is increased about 4-fold in HeLa cells by cycloheximide (CHX) or puromycin at concentrations that inhibit protein synthesis. This effect is observed in a number of cell lines that ectopically produce alpha-subunit, including ChaGo (brochogenic carcinoma), FL (amnion), and HeLa (cervical carcinoma). No increase in alpha-subunit mRNA is evident in two choriocarcinoma cell lines (JAr, JEG-3) that produce alpha-subunit as an eutopic product. The half-life of alpha-subunit mRNA is unchanged in the presence of CHX, but nuclear run-on assays demonstrate a 2.6-fold greater loading of RNA polymerase on the alpha-subunit gene in nuclei from CHX-treated cells. These results suggest that inhibition of protein synthesis results in higher transcription rates and not in decreased mRNA turnover. A nuclear protein (Mr 50,000) that binds to a DNA fragment located 5' proximal to the alpha-subunit gene but not to more distal 5'-flanking sequence or to the alpha-subunit cDNA has been identified in HeLa but not in JEG-3 cell lines. The p50 DNA binding activity in HeLa cells decreases in the presence of CHX at a rate similar to that at which alpha-subunit mRNA increases. Moreover, in a series of HeLa cell clones, the levels of p50 are directly proportional to the magnitude of induction produced by CHX. These data are consistent with a model for alpha-subunit gene regulation involving a labile repressor and constitute yet another level of differential regulation of the alpha-subunit gene in cells that produce the hormone subunit in an ectopic versus eutopic manner.

Cyclic AMP-dependent protein kinase regulates transcription of the phosphoenolpyruvate carboxykinase gene but not binding of nuclear factors to the cyclic AMP regulatory element.

We have examined the binding of factors in rat liver nuclear extracts to the phosphoenolpyruvate carboxykinase (PEPCK) gene cyclic AMP (cAMP) response element (CRE) and other CREs and have isolated a rat liver CRE-binding protein (CREBP) cDNA. In addition, we have examined the influence of altering the phosphorylation state of nuclear factors on both CRE binding and in vitro transcription. Specific binding to the PEPCK CRE was measured in a mobility shift assay. CRE sequences of the PEPCK, somatostatin, and glycoprotein hormone alpha subunit genes competed equally for binding of rat liver nuclear factors to the PEPCK CRE, whereas mutant PEPCK CRE sequences did not compete for binding. Oligonucleotides complementary to rat pheochromocytoma CREBP (Gonzalez et al., Nature [London] 337:749-752, 1989) were used to prime rat liver and brain cDNA in the polymerase chain reaction. The predominant CREBP molecule obtained was identical to the rat pheochromocytoma CREBP except for a 14-amino-acid deletion in the N-terminal half that was also present in a human placental cDNA (Hoeffler et al., Science 242:1430-1433, 1988). The regulation of transcription by cAMP was examined by coincubation of rat liver nuclear extract with the purified catalytic subunit of cAMP-dependent protein kinase (protein kinase A). Although binding to the CRE was unaffected, in vitro transcription directed by the PEPCK promoter was stimulated by catalytic subunit, and this effect was blocked by protein kinase inhibitor peptide. In contrast, when nuclear extract was coincubated with phosphatase, there was substantial inhibition of in vitro transcription directed by the PEPCK promoter, but there was no effect on binding to the CRE. The major effects of catalytic subunit were exerted through the CRE, but residual stimulation was evident in promoter fragments containing only the TATA element. These data suggest that factors are bound to the CRE at constitutively high levels and that their capacity for transcriptional activation is regulated by phosphorylation.

Cyclic AMP-Dependent Protein Kinase Regulates Transcription of the Phosphoenolpyruvate Carboxykinase Gene but Not Binding of Nuclear Factors to the Cyclic AMP Regulatory Element

We have examined the binding of factors in rat liver nuclear extracts to the phosphoenolpyruvate carboxykinase (PEPCK) gene cyclic AMP (cAMP) response element (CRE) and other CREs and have isolated a rat liver CRE-binding protein (CREBP) cDNA. In addition, we have examined the influence of altering the phosphorylation state of nuclear factors on both CRE binding and in vitro transcription. Specific binding to the PEPCK CRE was measured in a mobility shift assay. CRE sequences of the PEPCK, somatostatin, and glycoprotein hormone alpha subunit genes competed equally for binding of rat liver nuclear factors to the PEPCK CRE, whereas mutant PEPCK CRE sequences did not compete for binding. Oligonucleotides complementary to rat pheochromocytoma CREBP (Gonzalez et al., Nature [London] 337:749-752, 1989) were used to prime rat liver and brain cDNA in the polymerase chain reaction. The predominant CREBP molecule obtained was identical to the rat pheochromocytoma CREBP except for a 14-amino-acid deletion in the N-terminal half that was also present in a human placental cDNA (Hoeffler et al., Science 242:1430-1433, 1988). The regulation of transcription by cAMP was examined by coincubation of rat liver nuclear extract with the purified catalytic subunit of cAMP-dependent protein kinase (protein kinase A). Although binding to the CRE was unaffected, in vitro transcription directed by the PEPCK promoter was stimulated by catalytic subunit, and this effect was blocked by protein kinase inhibitor peptide. In contrast, when nuclear extract was coincubated with phosphatase, there was substantial inhibition of in vitro transcription directed by the PEPCK promoter, but there was no effect on binding to the CRE. The major effects of catalytic subunit were exerted through the CRE, but residual stimulation was evident in promoter fragments containing only the TATA element. These data suggest that factors are bound to the CRE at constitutively high levels and that their capacity for transcriptional activation is regulated by phosphorylation.

Identification ofCis-Acting Promoter Elements Important for Expression of the Mouse Glycoprotein Hormone α-Subunit Gene in Thyrotropes

The glycoprotein hormone alpha-subunit gene is expressed in a cell-specific manner in the anterior pituitary and placenta. Previous studies have shown that the region between -178 to -111 is indispensable for placental-specific expression of the human alpha-subunit gene. Using gene transfer techniques with chimeric luciferase plasmids, this report identifies regions of the mouse alpha-subunit promoter that are important for transcriptional activation in primary thyrotropic cells. Transient expression of a series of 5' flanking DNA deletions resulted in stepwise reductions of basal promoter activity between -480 to -417 (4-fold), -254 to -177 (5-fold), and -177 to -120 (3.5-fold). DNase-I protection analysis with nuclear extracts from thyrotropic tumor cells revealed specific protein-DNA interactions within each of these functionally defined regions. These were mapped to positions -474 to -452, -447 to -419, -213 to -170, and -158 to -101 within the 5' flanking region. In contrast, in mouse fibroblast L-cells no significant difference in alpha-subunit promoter activity was found by deleting the region from -480 to -177. However, a 3-fold decrease, similar to that found in primary thyrotropes, was found by deleting the region from -177 to -120. Further, a smaller region between -138 and -122 was the only area detected by the DNase-I protection assay using L-cell nuclear extracts. Thus, several cis-acting promoter elements located up-stream of position -177 are important for expression in thyrotropes. These elements also bind nuclear factors present in thyrotropes but not in nonpituitary fibroblasts and, therefore, differ from those mediating expression of the human alpha-subunit gene in the placenta.

Amplification of the Transcriptional Signal Mediated by the Tandem cAMP Response Elements of the Glycoprotein Hormone α-Subunit Gene Occurs through Several Distinct Mechanisms

cAMP stimulates transcription of the human glycoprotein hormone alpha-subunit gene in choriocarcinoma cells. Combined treatment with phorbol esters potentiates this effect. Tandem cAMP response elements (CREs) in the proximal 5'-flanking sequence mediate the effect of cAMP. In this report, we show that the CREs can also mediate the synergistic effect of phorbol esters. In addition to serving as an inducible cis-acting element, the two CREs act synergistically to increase basal transcription. We now provide direct evidence via equilibrium binding studies that tandem CREs bind their trans-acting factors cooperatively. However, the level of cooperativity is insufficient to explain the high degree of transcriptional synergism, suggesting that another element in the alpha-subunit promoter may be required for complete synergism. In support of this hypothesis, we show that synergism is drastically reduced when the CREs are removed from the context of their native promoter and linked to a heterologous promoter. Thus, amplification of the transcriptional signal mediated by the tandem CREs occurs through at least three distinct mechanisms. First, at the level of signal transduction by convergence of the A- and C-kinase pathways; second, through homotropic interactions of trans-acting factors binding to tandem CREs; and finally through heterotropic interaction of the two CREs with another, as yet, undefined cis-acting element(s) in the human alpha-subunit promoter.

Cell Lines of the Pituitary Gonadotrope Lineage Derived by Targeted Oncogenesis in Transgenic Mice

Study of the molecular and cellular biology of the gonadotropin hormones would be greatly facilitated by the availability of immortalized anterior pituitary gonadotrope cell lines. We directed expression of the simian virus-40 (SV40) T-antigen (Tag) oncogene to specific cells in the anterior pituitary of transgenic mice using the promoter/enhancer region from the human glycoprotein hormone alpha-subunit gene. Transgenic mice carrying this fusion gene developed anterior pituitary tumors. Clonal cell lines established from these tumors express the endogenous mouse alpha-subunit gene and synthesize and secrete alpha-subunit protein. However, they do not express beta-subunit genes. Alpha-subunit mRNA is induced by GnRH in a dose- and time-dependent manner, but is not regulated by TRH. Thus, we have targeted tumorigenesis in transgenic mice to anterior pituitary cells of the gonadotrope lineage to immortalize this specific endocrine cell while maintaining several highly differentiated functions unique to gonadotropes.

Expression of the glycoprotein hormone alpha-subunit gene in the placenta requires a functional cyclic AMP response element, whereas a different cis-acting element mediates pituitary-specific expression.

The single-copy gene encoding the alpha subunit of glycoprotein hormones is expressed in the pituitaries of all mammals and in the placentas of only primates and horses. We have systematically analyzed the promoter-regulatory elements of the human and bovine alpha-subunit genes to elucidate the molecular mechanisms underlying their divergent patterns of tissue-specific expression. This analysis entailed the use of transient expression assays in a chorionic gonadotropin-secreting human choriocarcinoma cell line, protein-DNA binding assays, and expression of chimeric forms of human or bovine alpha subunit genes in transgenic mice. From the results, we conclude that placental expression of the human alpha-subunit gene requires a functional cyclic AMP response element (CRE) that is present as a tandem repeat in the promoter-regulatory region. In contrast, the promoter-regulatory region of the bovine alpha-subunit gene, as well as of the rat and mouse genes, was found to contain a single CRE homolog that differed from its human counterpart by a single nucleotide. This difference substantially reduced the binding affinity of the bovine CRE homolog for the nuclear protein that bound to the human alpha CRE and thereby rendered the bovine alpha-subunit promoter inactive in human choriocarcinoma cells. However, conversion of the bovine alpha CRE homolog to an authentic alpha CRE restored activity to the bovine alpha-subunit promoter in choriocarcinoma cells. Similarly, a human but not a bovine alpha transgene was expressed in placenta in transgenic mice. Thus, placenta-specific expression of the human alpha-subunit gene may be the consequence of the recent evolution of a functional CRE. Expression of the human alpha transgene in mouse placenta further suggests that evolution of placenta-specific trans-acting factors preceded the appearance of this element. Finally, in contrast to their divergent patterns of placental expression, both the human and bovine alpha-subunit transgenes were expressed in mouse pituitary, indicating differences in the composition of the enhancers required for pituitary- and placenta-specific expression.

Regulation of the Transfected Human Glycoprotein Hormone α-Subunit Gene by Dexamethasone and Thyroid Hormone

A chimeric plasmid, (-1,500)h alpha CAT, containing approximately 1,500 bp of 5'-flanking DNA of the human glycoprotein hormone alpha-subunit gene directing the expression of the bacterial chloramphenicol acetyl transferase (CAT) gene, was transfected transiently into rat pituitary-derived GH3 cells. (-1,500)h alpha CAT expression was stimulated 5- to 20-fold by dexamethasone and 3- to 5-fold by 8-bromo-cAMP (8-Br-cAMP), and was inhibited by 50% by L-triiodothyronine (T3). Thus, suppression by T3 in this system was similar to that seen in pituitary thyrotropes. Induction of (-1,500)h alpha CAT expression by dexamethasone was antagonized by T3 but was unaffected by 8-Br-cAMP. However, T3 augmented the stimulation of (-1,500)h alpha CAT activity by 8-Br-cAMP. Deletants containing less than 346 bp of 5'-flanking alpha DNA showed a stepwise decrease in induction by dexamethasone, suggesting that multiple sequence elements located in this region are required for full induction of h alpha CAT activity. Deletion analysis also indicated that a thyroid hormone response element is located between 207 and 172 bp of the alpha gene transcriptional start site. Our finding of induction of alpha expression by dexamethasone in pituitary cells contrasts with the inhibition of alpha gene activity by glucocorticoids which has previously been shown in placental cells. Therefore, these data indicate that cell-type-specific factors play an important role in the modulation of alpha gene transcription.

Thyroid Hormone Regulation of the Rat Glycoprotein Hormone α-Subunit Gene Promoter Activity

Abstract An important physiological control of the glycoprotein hormone alpha-subunit is the negative feedback by thyroid hormones in the thyrotrope. A region of the rat glycoprotein hormone alpha-subunit gene that is involved in transcriptional regulation by thyroid hormone has been identified by transient transfection studies, and sequence-specific binding of the thyroid hormone receptor to a site within this region has been demonstrated. Deletion-mutation studies using plasmid expression vectors containing either 246, 170, or 80 base pairs of the 5'-flanking region of the rat alpha-subunit gene fused to the coding region of the bacterial chloramphenicol acetyltransferase gene demonstrate 3,5,3'-triiodo-L-thyronine (T3)-regulated expression in GH3 cells, a T3-responsive somatotrophic cell line. In order to investigate the possibility of thyroid hormone receptor interaction with this segment of the rat alpha-subunit gene, the binding of the thyroid hormone receptor to synthetic oligodeoxynucleotides was analyzed using an avidin-biotin complex DNA binding assay. An oligodeoxyribonucleotide representing a fragment of the alpha-subunit gene from -74 to -38, relative to the transcriptional start site, shows significant binding to [125I]T3-receptor complex present in nuclear extracts of GH3 cells. This fragment binds receptor to a degree similar to that seen with a fragment of the rat growth hormone gene which contains a putative thyroid hormone-responsive element. In addition, this fragment of the rat alpha-subunit gene binds to the in vitro synthesized human c-erbA beta protein, which has been identified as a member of the family of putative T3 receptors. These data demonstrate that a cis-active thyroid hormone-responsive element resides in the 5'-flanking region of the rat alpha-subunit gene and that the mechanism involved in the suppression of expression of this gene by T3 could involve specific binding of the thyroid hormone receptor to this region of the gene.

Expression and regulation of the pituitary- and placenta-specific human glycoprotein hormone alpha-subunit gene is restricted to the pituitary in transgenic mice.

Expression of the glycoprotein hormone alpha subunit occurs in both the pituitary and placenta in humans. However, this study found that expression of this subunit is restricted to the pituitary in mice. An interspecies analysis of human alpha-subunit gene regulation was undertaken, using the transgenic-mouse approach. In mice transgenic for a genomic clone containing the complete human alpha-subunit gene and several kilobases of 5'- and 3'-flanking sequences, cell-type-specific expression and hormonal regulation of the human alpha-subunit transgene occurred in the mouse pituitary, whereas no expression of the transgene was detectable in the mouse placenta. These findings provide strong evidence that a common trans-acting factor(s) regulates glycoprotein hormone alpha-subunit gene expression in the human and mouse pituitaries; however, this factor(s) or a unique factor(s), though functional in the human placenta, is either nonfunctional or absent in the mouse placenta.

Expression and Regulation of the Pituitary- and Placenta-Specific Human Glycoprotein Hormone Alpha-Subunit Gene Is Restricted to the Pituitary in Transgenic Mice

Expression of the glycoprotein hormone alpha subunit occurs in both the pituitary and placenta in humans. However, this study found that expression of this subunit is restricted to the pituitary in mice. An interspecies analysis of human alpha-subunit gene regulation was undertaken, using the transgenic-mouse approach. In mice transgenic for a genomic clone containing the complete human alpha-subunit gene and several kilobases of 5'- and 3'-flanking sequences, cell-type-specific expression and hormonal regulation of the human alpha-subunit transgene occurred in the mouse pituitary, whereas no expression of the transgene was detectable in the mouse placenta. These findings provide strong evidence that a common trans-acting factor(s) regulates glycoprotein hormone alpha-subunit gene expression in the human and mouse pituitaries; however, this factor(s) or a unique factor(s), though functional in the human placenta, is either nonfunctional or absent in the mouse placenta.

Negative regulation by glucocorticoids through interference with a cAMP responsive enhancer.

Although steroid hormone receptors are known to activate gene expression by binding to specific hormone-dependent enhancers, the mechanisms by which steroids inhibit the transcription of specific genes are unknown. It is shown here by gene transfer studies that the same glucocorticoid receptor that activates gene expression can negatively regulate expression of the human glycoprotein hormone alpha-subunit gene. Glucocorticoid inhibition was conferred by a 52-nucleotide region that also contains elements crucial both for adenosine 3',5'-monophosphate (cAMP) responsiveness and for placental-specific expression of this gene and was observed only under conditions in which these elements were functioning as enhancers. Purified glucocorticoid receptor was found to bind to DNA that overlap the cAMP responsive elements sites in this region. It is hypothesized that steroid receptors negatively regulate gene expression by interfering with the activity or binding of other important transcription factors.

Tissue-specific enhancer of the human glycoprotein hormone alpha-subunit gene: dependence on cyclic AMP-inducible elements.

We identified and characterized elements which confer tissue specificity and cyclic AMP (cAMP) responsiveness to the human glycoprotein alpha-subunit gene. An enhancer containing an 18-base-pair repeat conferred cAMP responsiveness in a non-tissue-specific fashion. DNase I protection assays revealed DNA-binding factors that bound to this element in both placental and nonplacental cells. It also enhanced the alpha-subunit promoter in a tissue-specific manner but had a negligible effect on a heterologous promoter. A unique element found upstream of this enhancer had no independent activity but, in combination with the cAMP-responsive enhancer, distinctly increased the tissue-specific activity of both the alpha-subunit promoter and a heterologous promoter. A factor that bound to this upstream element was found in placental but not nonplacental cells. We conclude that this novel element acts, perhaps through a specific trans-acting factor, in concert with a cAMP-responsive enhancer to confer tissue specificity to the alpha-subunit gene.

Cyclic AMP regulation of the human glycoprotein hormone alpha-subunit gene is mediated by an 18-base-pair element.

cAMP regulates transcription of the gene encoding the alpha-subunit of human chorionic gonadotropin (hCG) in choriocarcinoma cells (BeWo). To define the sequences required for regulation by cAMP, we inserted fragments from the 5' flanking region of the alpha-subunit gene into a test vector containing the simian virus 40 early promoter (devoid of its enhancer) linked to the bacterial chloramphenicol acetyltransferase (CAT) gene. Results from transient expression assays in BeWo cells indicated that a 1500-base-pair (bp) fragment conferred cAMP responsiveness on the CAT gene regardless of position or orientation of the insert relative to the viral promoter. A subfragment extending from position -169 to position -100 had the same effect on cAMP-induced expression. Furthermore, the entire stimulatory effect could be achieved with an 18-bp synthetic oligodeoxynucleotide corresponding to a direct repeat between positions -146 and -111. In the absence of cAMP, the alpha-subunit 5' flanking sequence also enhanced transcription from the simian virus 40 early promoter. We localized this enhancer activity to the same -169/-100 fragment containing the cAMP response element. The 18-bp element alone, however, had no effect on basal expression. Thus, this short DNA sequence serves as a cAMP response element and also functions independently of other promoter-regulatory elements located in the 5' flanking sequence of the alpha-subunit gene.