The CGTCA sequence motif is essential for biological activity of the vasoactive intestinal peptide gene cAMP-regulated enhancer.

Abstract

cAMP-regulated transcription of the human vasoactive intestinal peptide gene is dependent upon a 17-basepair DNA element located 70 base pairs upstream from the transcriptional initiation site. This element is similar to sequences in other genes known to be regulated by cAMP and to sequences in several viral enhancers. We have demonstrated that the vasoactive intestinal peptide regulatory element is an enhancer that depends upon the integrity of two CGTCA sequence motifs for biological activity. Mutations in either of the CGTCA motifs diminish the ability of the element to respond to cAMP. Enhancers containing the CGTCA motif from the somatostatin and adenovirus genes compete for binding of nuclear proteins from C6 glioma and PC12 cells to the vasoactive intestinal peptide enhancer, suggesting that CGTCA-containing enhancers interact with similar transacting factors.DNA control elements that activate transcription in response to intracellular effectors such as cAMP or products of phosphoinositol metabolism have been identified in several cellular and viral genes (1-6). These genetic elements may be particularly important in neuroendocrine cells where the activation of intracellular second messenger pathways may couple secretion and biosynthesis of neuropeptides (7,8). The genetic element responsible for cAMP-regulated transcription of the human vasoactive intestinal peptide (VIP) gene consists ofa 17-base-pair (bp) sequence located between 70 and 86 bp upstream from the transcriptional initiation site (5). This sequence in the VIP gene is similar to the cAMPresponsive elements (CREs) of the rat somatostatin, human proenkephalin, human a-chorionic gonadotrophin, and rat phosphoenolpyruvate carboxykinase genes (1-5). All of these CREs contain one or multiple copies of the conserved sequence motif CGTCA and several have been shown to have properties of enhancers (2-4). The CGTCA motif is also found in several viral enhancers such as the 18-bp repeat of the cytomegalovirus gene and the ElA-inducible element of the adenovirus E4 promoter (9, 10). The presence of the CGTCA motif in these viral enhancers suggests that similar nuclear factors may activate transcription of both viral and cellular genes. In the VIP CRE, the two CGTCA motifs are present as inverted repeats, a structure characteristic of some other enhancers (11-13). It is not known whether the palindromic structure of the VIP CRE is necessary for biological activity.In this study, three major issues were addressed. First, we sought to determine whether the VIP CRE could be categorized as an enhancer. Second, we examined the importance of the CGTCA motifs for activity of the DNA element.

Regulation of the NRSF/REST gene by methylation and CREB affects the cellular phenotype of small-cell lung cancer

“…Many promoters of cAMP-responsive genes only contain a half-site TGACG sequence (Barco and Kandel, 2006), which is essential for biological activity (Fink et al, 1988). This same sequence found here in reverse orientation was shown to be a functional element.…”

Internalization of the Human Nicotinic Acid Receptor GPR109A Is Regulated by Gi, GRK2, and Arrestin3

“…The SV40 early mRNA polyadenylation signal was cloned from the pEGFP-N1 vector using the forward primer (5Ј-CTC GAG CAT AAT CAG CCA TAC CAC ATT TGT A-3Ј) and reverse primer (5Ј-GGT ACC GGA CAA ACC ACA ACT AGA ATG CA-3Ј); XhoI and KpnI sites were incorporated into the 5Ј-and 3Ј-ends. The cAMP-response element (CRE) was synthesized based on a previously published sequence (19,20) and inserted into the pBluescript KS II(ϩ) (Stratagene, La Jolla, CA) vector, followed by a minipromoter VIP, firefly luciferase, and SV40 poly(A) signal.…”

Modulation of tax and PKA‐mediated expression of HTLV‐I promoter via cAMP response element binding and modulator proteins CREB and CREM

“…The palindromic CRE sequence (TGACGTCA) exemplified by COL8 [19] is composed of two functional CGTCA units essential for cAMP response [31] which overlap in opposite DNA strands, forming a symmetrical CRE (Table 1). In contrast, CREs in the 21-bp repeats of HTLV-I (H21CRE) fall into the second category of low affinity sites represented by asymmetric CREs (Table I).…”