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

Abstract

ABSTRACTcAMP regulates transcription of the gene encoding the a-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 a-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 a-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 a-subunit gene.Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone expressed in the placenta. Both the a-subunit and the P-subunit are required for biological activity (1). While a physiological regulator of hCG production has not been identified, the synthesis of both subunits can be stimulated by cAMP in placental explants and in human choriocarcinoma cells (2, 3). Recent reports from several laboratories have shown that cAMP regulates expression of the chorionic gonadotropin a-and ,-subunit genes, at least in part, at the level of transcription (refs. 4 and 5; A.M., R. Cox, and J.H.N., unpublished data).In the human a-subunit gene, the first 140 base pairs (bp) of 5' flanking sequence are sufficient to confer cAMP regulation to a heterologous gene after transfection and transient expression in choriocarcinoma cells (4). This suggests that a cAMP response element lies within this region. In the present study, we have constructed several expression vectors and have used a transient expression assay to localize this element to an 18-bp sequence that is repeated between positions -146 and -111 in the 5' flanking region of the a-subunit gene. A single copy ofthis cAMP response element is sufficient to confer the same degree of cAMP regulation as a 1500-bp fragment containing the a-subunit promoter. This response element functions independently of other promoter regulatory elements. PROCEDURES Construction of Vectors. Construction of the expression vector pHaCAT (Fig. 1A) was initiated by isolating a 1500-bp DNA fragment from the genomic clone of the human asubunit gene provided by J. Fiddes (6). ...

Identification of basal and cyclic AMP regulatory elements in the promoter of the phosphoenolpyruvate carboxykinase gene.

“…This effect on basal promoter activity of the aCG gene CRE was restricted to choriocarcinoma cells and required a tandem repeat of the CRE consensus sequence (6). In contrast, Silver et al (43) found no effect of the 18-bp repeat of the aCG gene on basal promoter activity of a different choriocarcinoma cell line but did observe significant stimulation when longer 5'-flanking sequences of the aCG gene were fused to the SV1 promoter in a CAT fusion gene.…”

“…The CREs of PEPCK and other genes have the properties of enhancers; they act in a relatively distance-and orientation-independent manner on their own promoters as well as on heterologous promoters (5,6,33,43,44). However, the core consensus sequence of the somatostatin gene was shown to be insufficient for mediation of cAMP responses (33), which indicates that additional sequence is required.…”

“…Previous reports have demonstrated the existence of cAMP regulatory elements in the 5'-flanking regions of the genes encoding PEPCK (42), somatostatin (33), proenkephalin (5), vasoactive intestinal peptide (44), and the alpha subunit of chorionic gonadotropin (aCG) (6,43). The CREs of PEPCK and other genes have the properties of enhancers; they act in a relatively distance-and orientation-independent manner on their own promoters as well as on heterologous promoters (5,6,33,43,44).…”

A CRE/ATF-like site in the upstream regulatory sequence of the human interleukin 1 beta gene is necessary for induction in U937 and THP-1 monocytic cell lines.

“…It is clear, however, that flanking sequence can play an important role in influencing the level of activation and tissue specificity. The actual size of the CRE/ATF sites can vary a great deal in the number of bases required for proper activity (10,32,38,40). Our observations that part of the NF-KB-like site is protected from Exo III digestion and that mutation of the NF-KB-like site results in an increase in induction of the IL-13-CAT construct in U937 cells may mean that the actual CRE/ATF-like site includes the NF-KB-like site.…”

The 5′‐flanking region of the human P‐450(SCC) gene shows responsiveness to CAMP‐dependent regulation in a transient gene‐expression system of Y‐1 adrenal tumor cells

“…Recently the CAMP-responsive DNA elements of several genes, including rat phosphoenolpyruvate carboxykinase [22], somatostatin [23], human proenkephalin [24] and chorionic gonadotropin genes [25], have been reported to have some homologous sequence in common. Reverse genetic analysis of the bovine P-450(C21) gene, another steroidogenic P-450 gene, has been performed with Y-1 adrenal cells to clarify a regulatory region of the gene expression by Parker et al [26].…”