Enhancer and promoter element interactions dictate cyclic adenosine monophosphate mediated and cell-specific expression of the glycoprotein hormone alpha-gene

The glycoprotein hormone alpha-gene is preferentially expressed in placental cell lines, but it is also expressed in several other cell lines indicating that the differential activity of the alpha-gene regulatory elements in various cell types is more quantitative than qualitative. The 5'-flanking region of the alpha-gene contains several distinct DNA regulatory sequences including an upstream regulatory element [(URE) -181 to -150 base pairs (bp)] that stimulates basal expression and an 18 bp twice-repeated cAMP-responsive element [(CRE) -146 to -111 bp]. We constructed an array of fusion genes containing the URE and/or the CRE linked to different truncated promoters [alpha-gene, somatostatin (SRIF), glucagon, Simian Virus 40]. These constructions were transiently expressed in placental, fibroblast, or islet cell lines to identify regulatory sequences involved in cell-specific expression as well as interactions between the URE, the CRE, and different promoter elements. The URE, CRE, and alpha-promoter elements contribute approximately 3-, 6-, and 5-fold, respectively, to preferential expression in JEG-3 cells. In JEG-3 cells, the URE is strictly dependent on the CRE for activity, but it functions in a promoter-independent manner. In contrast, the CRE is markedly promoter dependent. When linked to heterologous enhancers, the alpha-promoter is more active in JEG-3 cells than in other cell lines, thereby contributing substantially to preferential expression in placental cells. Although the CREs derived from the alpha and SRIF genes both activate expression of the alpha promoter, only the alpha CRE activates the SRIF promoter in JEG-3 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tissue-specific regulation of the ecto-5'-nucleotidase promoter. Role of the camp response element site in mediating repression by the upstream regulatory region.

We have isolated the 5' region of the ecto-5'-nucleotidase (low K(m) 5'-NT) gene and established that a 969-base pair (bp) fragment confers cell-specific expression of a CAT reporter gene that correlates with the expression of endogenous ecto-5'-NT mRNA and enzymatic activity. A 768-bp upstream negative regulatory region has been identified that conferred lymphocyte-specific negative regulation in a heterologous system with a 244-bp deoxycytidine kinase core promoter. DNase I footprinting identified several protected areas including Sp1, Sp1/AP-2, and cAMP response element (CRE) binding sites within the 201-bp core promoter region and Sp1, NRE-2a, TCF-1/LEF-1, and Sp1/NF-AT binding sites in the upstream regulatory region. Whereas the CRE site was essential in mediating the negative activity of the upstream regulatory region in Jurkat but not in HeLa cells, mutation of the Sp1/AP-2 site decreased promoter activity in both cell lines. Electrophoretic mobility shift assay analysis of proteins binding to the CRE site identified both ATF-1 and ATF-2 in Jurkat cells. Finally, phorbol 12-myristate 13-acetate increased the activity of both the core and the 969-bp promoter fragments, and this increase was abrogated by mutations at the CRE site. In summary, we have identified a tissue-specific regulatory region 5' of the ecto-5'-NT core promoter that requires the presence of a functional CRE site within the basal promoter for its suppressive activity.     

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

Promoter elements important for basal and cyclic AMP (cAMP)-regulated expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene have been identified by analysis of a series of PEPCK promoter mutations in transfection experiments. Fusion genes containing wild-type and mutated PEPCK promoter sequences from -600 to +69 base pairs (bp) fused to the coding sequence for chloramphenicol acetyltransferase were studied. Internal deletion mutations that replaced specific bases with a 10-bp linker within the region from -129 bp to -18 bp of the PEPCK promoter were examined. In addition, wild-type and mutated DNA templates were used as probes in DNase I protection experiments to determine sites of protein-DNA interaction. The PEPCK promoter contains a binding site for nuclear factor 1-CAAT. Deletion of the 5' end of this binding site reduced the size of the DNase I footprint in this region but had no effect on promoter activity. In contrast, deletion or disruption of the 3' end of this binding site completely eliminated protein binding and reduced promoter activity by 50%. Deletion of core sequences of the cAMP regulatory element (CRE) resulted in loss of cAMP responsiveness and an 85% decrease in basal promoter activity, indicating that the CRE also functions as a basal stimulatory element. Mutation of the core sequence of the CRE resulted in loss of the DNase I footprint over the CRE. Internal deletions flanking the CRE showed no loss of induction by cAMP but did have reduced promoter activity. This delimits the CRE to an 18-bp region between nucleotides -100 and -82. Analysis of mutations that disrupted bases between the CRE and the initiation site identified a basal inhibitory element adjacent to a basal stimulatory element, both located just 3' of the CRE, as well as a basal stimulatory element coincident with the TATA consensus sequence centered at -27. These data demonstrate that several cis-acting elements are located within 130 nucleotides of the initiation site of the PEPCK gene and that the CRE is essential for both basal promoter activity and cAMP-regulated expression of this gene.     

Transcriptional activation of the rat glucagon gene by the cyclic AMP-responsive element in pancreatic islet cells.

The 5'-flanking region of the rat glucagon gene contains, from nucleotides -291 to -298, a sequence (TGA CGTCA) which mediates cyclic AMP (cAMP) responsiveness in several genes (cAMP-responsive element [CRE]). However, because of nonpermissive bases surrounding the CRE octamer, the glucagon CRE does not confer cAMP responsiveness to an inert heterologous promoter in placental JEG cells that do not express the glucagon gene. This report describes transient transfection experiments with glucagon-reporter fusion genes that show that glucagon gene expression is activated by cAMP-dependent protein kinase A in a glucagon-expressing pancreatic islet cell line. This activation is mediated through the glucagon CRE.     

Transcriptional control of the mouse prealbumin (transthyretin) gene: both promoter sequences and a distinct enhancer are cell specific.

The mouse genomic clone for the prealbumin (transthyretin) gene was cloned, and its upstream regulatory regions were analyzed. The 200 nucleotides 5' to the cap site when placed within a recombinant plasmid were sufficient to direct transient expression in HepG2 (human hepatoma) cells, but this DNA region did not support expression in HeLa cells. The sequence of the 200-nucleotide region is highly conserved between mouse and human DNA and can be considered a cell-specific promoter. Deletions of this promoter region identified a crucial element for cell-specific expression between 151 and 110 nucleotides 5' to the RNA start site. A region situated at about 1.6 to 2.15 kilobases upstream of the RNA start site was found to stimulate expression 10-fold in HepG2 cells but not in HeLa cells. This far upstream element was invertible and increased expression from the beta-globin promoter in HepG2 cells. Unlike the simian virus 40 enhancer, the prealbumin enhancer would not stimulate beta-globin synthesis in HeLa cells, and even the simian virus 40 enhancer did not stimulate the prealbumin promoter in HeLa cells. Thus, we identified in the prealbumin gene two DNA elements that respond in a cell-specific manner: a proximal promoter including a crucial sequence between -108 and -151 nucleotides and a distant enhancer element located between 1.6 and 2.15 kilobases upstream.     

Characterization of promoter elements required for cell-specific expression of the neurotensin/neuromedin N gene in a human endocrine cell line.

Expression of the gene encoding neurotensin/neuromedin N (NT/N) is mostly limited to the brain and specialized enteroendocrine cells (N cells) of the distal small intestine. We have analyzed the NT/N DNA sequences upstream of the RNA start site that direct cell-specific expression using a novel human endocrine cell line, BON, that resembles intestinal N cells in several important aspects, including NT/N precursor protein processing, ratios of different NT/N mRNA forms, and high levels of constitutive expression of the NT/N gene. Transient transfection assays with plasmids with progressive 5' deletions of the rat NT/N promoter identified the proximal 216 bp of 5' flanking sequences as essential for high-level constitutive NT/N expression in BON cells. In addition, a detailed mutational analysis defined multiple regions within the proximal 216 bp that contribute to cell-specific NT/N expression. These elements include a proximal cyclic AMP response element (CRE)/AP-1-like motif (TGACATCA) that binds c-Jun, JunD, CRE-binding (CREB), and ATF proteins, a near-consensus glucocorticoid response element, and a distal consensus AP-1 site that binds c-Fos, Fra-1, and JunD. In addition, elements contained within two 21-bp imperfect direct repeats play an important role in NT/N expression in BON cells and may bind novel factors that act as positive regulators of NT/N expression. DNase I footprinting and gel shift analyses demonstrate that the sites identified by mutational analysis, and at least one additional site, specifically bind BON cell nuclear proteins in vitro. We speculate that a complex pattern of regulation requiring interaction between a proximal CRE/AP-1-like motif and other upstream control elements play an important role in the high-level constitutive expression of NT/N in the human endocrine cell line BON. In addition, the BON cell line provides a unique model to further characterize the factors regulating cell-specific NT/N expression and to better understand the mechanisms responsible for the terminal differentiation of the N-cell lineage in the gut.     

A cAMP-responsive element regulates expression of the mouse steroid 11 beta-hydroxylase gene

In Y1 mouse adrenocortical tumor cells, expression of steroid 11 beta-hydroxylase (11 beta-OHase) is stimulated by cAMP following a delay of 4-6 h. Our results demonstrate that a cAMP-responsive element (CRE) within the 11 beta-OHase promoter region is a major determinant of this induction. The 5'-flanking sequences from the mouse 11 beta-OHase gene were placed in front of a human growth hormone reporter gene and transfected into Y1 cells. Treatment of transfected cells with 8-bromo-cAMP increased expression directed by the 11 beta-OHase 5'-flanking region by 3.8-fold. In 5'-deletion analyses, 123 base pairs of 5'-flanking sequences were sufficient for cAMP induction, whereas cAMP treatment did not affect expression of a plasmid with only 40 base pairs of 5'-flanking sequence. Within these 123 base pairs, a region from -56 to -49 matched 7 of 8 bases comprising the consensus sequence for the CRE. 11 beta-OHase 5'-flanking sequences from -65 to -42, including the CRE-like sequence, conferred cAMP inducibility to promoters from the thymidine kinase and chorionic gonadotropin alpha-subunit genes. DNase I footprinting and Southwestern blotting analyses demonstrated that the protein which interacted with the CRE in the 11 beta-OHase promoter region was similar to the CRE-binding protein associated with other cAMP-regulated genes. Together, these results suggest that an interaction between the 11 beta-OHase CRE and CRE-binding protein mediates cAMP induction of the 11 beta-OHase gene.     

Structure of the murine mb-1 gene encoding a putative sIgM-associated molecule

Genomic DNA clones containing the B cell-specific murine mb-1 gene were isolated and a 5.6-kb BamH I fragment was characterized. It is 5629 bp long and contains five exons: an exon containing the 5' untranslated and the coding sequence of the signal peptide, an exon of 294 bp, which contains most of the extracellular sequence of the MB-1 protein, a 119-bp long exon coding mainly for the transmembrane portion, and two exons of 69 bp and 427 bp encoding the cytoplasmic domain and the 3'-untranslated region, respectively. The mb-1 gene does not contain a "TATA box" found in many eukaryotic promoters. The 5'-flanking region has sequence stretches homologous to IgVH 5'-promoter regions and a bcl 2 intron sequence. It contains the decanucleotide sequence (ATGGCAAATA) almost identical to the octamer motif of IgVH promoters. A B cell-specific DNase I-hypersensitive site was found in the 3'-flanking region indicating that this region might be involved in B cell-specific expression of mb-1. Southern blot analysis of genomic liver DNA with the cloned mb-1 cDNA suggests the existence of another mb-1-related gene segment.