Novel Cyclic Adenosine 3',5'Monophosphate Response Element in the Human Chorionic Gonadotropin /?-Subunit Gene
Christopher Albanese, Thomas W. H. Kay, Noreen M. Troccoli, and J. Larry Jameson
CG is encoded by separate a- and /?-subunit genes. Expression of both genes is stimulated by cAMP, but the kinetics of activation are different, with cAMP stimulation of the a gene preceding that of the 0 gene. The cAMP response element (CRE) in the a gene contains a palindromic DNA sequence, TGACGTCA, that binds the transcription factor CREB, a nuclear phosphoprotein that is activated by protein kinase-A. Previously, detailed characterization of a CRE in the CG/? gene had been difficult due to low levels of expression in transfected cells. In this study the 5'-flanking sequence of the CG/? gene was fused to a sensitive luciferase (LUC) reporter gene, allowing delineation of a CG/? CRE in transient expression assays performed in JEG-3 choriocarcinoma cells. The full-length CG/? promoter, -3700 to 362 basepairs (bp), was stimulated 8- to 14-fold by treatment with 1 miui 8-bromo-cAMP. Analyses of a series of deletion mutants in the CG/? promoter demonstrated that - 3 1 1 CG/3LUC retained nearly complete cAMP stimulation, but deletion to -187 bp eliminated cAMP responsiveness. Overlapping DNA fragments between - 3 1 1 and - 3 0 bp were fused to a heterologous promoter (-99aLUC) to further define the locations of basal elements and CREs. Basal expression required a combination of at least two distinct elements between - 3 1 1 and - 3 0 bp, whereas cAMP responsiveness was conferred by sequences between - 3 1 1 and - 2 0 2 bp. Shorter DNA sequences within this region were insufficient for cAMP stimulation, suggesting that more than one element may be required. DNase-l footprinting and gel mobility shift studies demonstrated at least three distinct protein-binding sites within the CG/? CRE sequence. Recombinant CREB (expressed in £. coli) did not bind to these sites, and they share no sequence homology with the a gene CRE, indicating that a cAMP-responsive transcription factor other
than CREB interacts with the CG/? promoter. (Molecular Endocrinology 5: 693-702, 1991)
INTRODUCTION
Cyclic AMP regulates many cellular processes, including patterns of gene expression. The effects of cAMP on gene expression are complex and have been demonstrated to involve transcriptional stimulation (1) and repression (2, 3) as well as mRNA stability (4-8). Analyses of the kinetics of cAMP stimulation in different systems suggest that multiple pathways and transcription factors may be involved in cAMP-stimulated transcription (1). In some instances, treatment with cAMP leads to rapid transcriptional stimulation, which is not inhibited by treatment with cycloheximide (1, 7, 9). This type of pathway probably involves the phosphorylation of preexisting transcription factors that mediate transcriptional activation (10, 11). In other cases, rapid activation of gene expression by cAMP is followed by repression (12, 13), consistent with a mechanism in which a secondary process subsequently inhibits cAMP-stimulated transcription. Finally, some genes are stimulated by cAMP with delayed kinetics, and their activation is either partially or completely blocked by treatment with cycloheximide, implying that indirect mechanisms involving protein synthesis may be required for induction of these genes (1, 7, 9, 14). Because of the complexity of transcriptional activation and the relatively large number of proteins that are typically involved, analysis of the cAMP-stimulated process is greatly facilitated by defining specific cAMP-responsive sequences. Despite the many known cAMP-responsive genes, only a few such elements have been well characterized (1). One of the first cAMP response elements (CREs) to be described in eukaryotes was the palindromic sequence TGACGTCA, which was identified in the rat somatostatin gene (15) and the hCG a-subunit gene
0888-8809/91 /0693-0702$03.00/0 Molecular Endocrinology Copyright © 1991 by The Endocrine Society
693
Downloaded from https://academic.oup.com/mend/article-abstract/5/5/693/2714405 by guest on 11 January 2019
Thyroid Unit Massachusetts General Hospital Harvard Medical School Boston, Massachusetts 02114
Vol 5 No. 5
MOL ENDO-1991 694
RESULTS
of the reporter assay system. The a and CG/3 promoters were, therefore, linked to the more sensitive luciferase (LUC) reporter gene for the current studies. Although transfected aLUC activity is maximal using 2 ng plasmid, dose-response analyses of transfected CG/3LUC plasmid (0-30 ^g) showed a linear increase in nuclear plasmid DNA and luciferase activity (data not shown). These features allowed the use of a relatively large amount of CG/3LUC plasmid (30 ng), providing a further increase in the level of reporter gene activity. The kinetics of cAMP induction were reevaluated using aLUC and CG0LUC (Fig. 1). Basal «LUC expression was approximately 50-fold greater than that of CG/3LUC. However, owing to the increased sensitivity of the CG/3LUC reporter gene, the level of basal CG0 expression (52,500 light units) was readily measured relative to background levels of a promoterless plasmid such as pOLUC (3,050 light units). The kinetics of cAMP induction of aLUC were rapid, with increased expression occurring as early as 30 min after the addition of 8-bromo-cAMP. In contrast, CG/3LUC expression was not significantly increased until 3 h after treatment with 8-bromo-cAMP. This experiment confirms the delay in cAMP stimulation of the CG/3 promoter and demonstrates that the CG/JLUC reporter gene provides considerably greater sensitivity than CG/3CAT reporter genes, thereby allowing mutational analyses of putative CREs. Delineation of Basal and cAMP Regulatory Elements in the Native CG# Promoter A series of 5' deletion mutants of the CGjSLUC reporter plasmid were transfected into JEG-3 cells to localize basal and cAMP regulatory elements. There was a marked drop in basal activity upon deletion between -3700 and -775 basepairs (bp) (Fig. 2A). However, there was little alteration in the level of basal expression with further deletions between -775 and - 8 7 bp. In parallel, groups of transfected cells were also analyzed
Christopher Albanese, Thomas W. H. Kay, Noreen M. Troccoli, and J. Larry Jameson
CG is encoded by separate a- and /?-subunit genes. Expression of both genes is stimulated by cAMP, but the kinetics of activation are different, with cAMP stimulation of the a gene preceding that of the 0 gene. The cAMP response element (CRE) in the a gene contains a palindromic DNA sequence, TGACGTCA, that binds the transcription factor CREB, a nuclear phosphoprotein that is activated by protein kinase-A. Previously, detailed characterization of a CRE in the CG/? gene had been difficult due to low levels of expression in transfected cells. In this study the 5'-flanking sequence of the CG/? gene was fused to a sensitive luciferase (LUC) reporter gene, allowing delineation of a CG/? CRE in transient expression assays performed in JEG-3 choriocarcinoma cells. The full-length CG/? promoter, -3700 to 362 basepairs (bp), was stimulated 8- to 14-fold by treatment with 1 miui 8-bromo-cAMP. Analyses of a series of deletion mutants in the CG/? promoter demonstrated that - 3 1 1 CG/3LUC retained nearly complete cAMP stimulation, but deletion to -187 bp eliminated cAMP responsiveness. Overlapping DNA fragments between - 3 1 1 and - 3 0 bp were fused to a heterologous promoter (-99aLUC) to further define the locations of basal elements and CREs. Basal expression required a combination of at least two distinct elements between - 3 1 1 and - 3 0 bp, whereas cAMP responsiveness was conferred by sequences between - 3 1 1 and - 2 0 2 bp. Shorter DNA sequences within this region were insufficient for cAMP stimulation, suggesting that more than one element may be required. DNase-l footprinting and gel mobility shift studies demonstrated at least three distinct protein-binding sites within the CG/? CRE sequence. Recombinant CREB (expressed in £. coli) did not bind to these sites, and they share no sequence homology with the a gene CRE, indicating that a cAMP-responsive transcription factor other
than CREB interacts with the CG/? promoter. (Molecular Endocrinology 5: 693-702, 1991)
INTRODUCTION
Cyclic AMP regulates many cellular processes, including patterns of gene expression. The effects of cAMP on gene expression are complex and have been demonstrated to involve transcriptional stimulation (1) and repression (2, 3) as well as mRNA stability (4-8). Analyses of the kinetics of cAMP stimulation in different systems suggest that multiple pathways and transcription factors may be involved in cAMP-stimulated transcription (1). In some instances, treatment with cAMP leads to rapid transcriptional stimulation, which is not inhibited by treatment with cycloheximide (1, 7, 9). This type of pathway probably involves the phosphorylation of preexisting transcription factors that mediate transcriptional activation (10, 11). In other cases, rapid activation of gene expression by cAMP is followed by repression (12, 13), consistent with a mechanism in which a secondary process subsequently inhibits cAMP-stimulated transcription. Finally, some genes are stimulated by cAMP with delayed kinetics, and their activation is either partially or completely blocked by treatment with cycloheximide, implying that indirect mechanisms involving protein synthesis may be required for induction of these genes (1, 7, 9, 14). Because of the complexity of transcriptional activation and the relatively large number of proteins that are typically involved, analysis of the cAMP-stimulated process is greatly facilitated by defining specific cAMP-responsive sequences. Despite the many known cAMP-responsive genes, only a few such elements have been well characterized (1). One of the first cAMP response elements (CREs) to be described in eukaryotes was the palindromic sequence TGACGTCA, which was identified in the rat somatostatin gene (15) and the hCG a-subunit gene
0888-8809/91 /0693-0702$03.00/0 Molecular Endocrinology Copyright © 1991 by The Endocrine Society
693
Downloaded from https://academic.oup.com/mend/article-abstract/5/5/693/2714405 by guest on 11 January 2019
Thyroid Unit Massachusetts General Hospital Harvard Medical School Boston, Massachusetts 02114
Vol 5 No. 5
MOL ENDO-1991 694
RESULTS
of the reporter assay system. The a and CG/3 promoters were, therefore, linked to the more sensitive luciferase (LUC) reporter gene for the current studies. Although transfected aLUC activity is maximal using 2 ng plasmid, dose-response analyses of transfected CG/3LUC plasmid (0-30 ^g) showed a linear increase in nuclear plasmid DNA and luciferase activity (data not shown). These features allowed the use of a relatively large amount of CG/3LUC plasmid (30 ng), providing a further increase in the level of reporter gene activity. The kinetics of cAMP induction were reevaluated using aLUC and CG0LUC (Fig. 1). Basal «LUC expression was approximately 50-fold greater than that of CG/3LUC. However, owing to the increased sensitivity of the CG/3LUC reporter gene, the level of basal CG0 expression (52,500 light units) was readily measured relative to background levels of a promoterless plasmid such as pOLUC (3,050 light units). The kinetics of cAMP induction of aLUC were rapid, with increased expression occurring as early as 30 min after the addition of 8-bromo-cAMP. In contrast, CG/3LUC expression was not significantly increased until 3 h after treatment with 8-bromo-cAMP. This experiment confirms the delay in cAMP stimulation of the CG/3 promoter and demonstrates that the CG/JLUC reporter gene provides considerably greater sensitivity than CG/3CAT reporter genes, thereby allowing mutational analyses of putative CREs. Delineation of Basal and cAMP Regulatory Elements in the Native CG# Promoter A series of 5' deletion mutants of the CGjSLUC reporter plasmid were transfected into JEG-3 cells to localize basal and cAMP regulatory elements. There was a marked drop in basal activity upon deletion between -3700 and -775 basepairs (bp) (Fig. 2A). However, there was little alteration in the level of basal expression with further deletions between -775 and - 8 7 bp. In parallel, groups of transfected cells were also analyzed
