cAMP Response Element Sequence Research Articles

Presence of cyclic AMP response element-binding protein in oligodendrocytes.

Several lines of evidence indicate that cyclic AMP (cAMP) induces oligodendrocytes differentiation. However, the mechanism(s) of this stimulation remains unknown. Because in several cell types the transcriptional activity of various cAMP-responsive genes is regulated through a cis-acting DNA sequence known as cAMP response element (CRE), we investigated the possible presence of a CRE binding (CREB) protein in myelinating oligodendrocytes. A double-stranded oligonucleotide containing a tandem repeat of the CRE sequence was labeled with T4 kinase in the presence of [32P]ATP and then incubated with a nuclear protein extract from 14-day-old rat brain oligodendrocytes. The reaction mixture was then electrophoresed on nondenaturing polyacrylamide gels. The results indicated the presence of a protein that specifically binds to the CRE sequence. The results were supported by southwestern blotting assays in which the CRE probe bound to a approximately 45-kDa protein species. In separate experiments, it was shown that the 45-kDa protein can be phosphorylated in vitro by the catalytic subunit of protein kinase A. Developmental analysis of CREB protein expression indicated a peak at 14 days of age, preceding the peak of myelinogenesis.

Characterization of the promoter-regulatory region and structural organization of E1 alpha gene (BCKDHA) of human branched-chain alpha-keto acid dehydrogenase complex.

We have isolated genomic clones containing the complete exon 1 and the promoter-regulatory region of the E1 alpha gene (BCKDHA) of human branched-chain alpha-keto acid dehydrogenase complex. The cloning was achieved by amplification of a genomic library in the SRB (P2) host strain that allowed the replication of nonstandard DNA structures. The results of this and previous (Dariush, N., Fisher, C. W., Cox, R. P., and Chuang, D. T. (1991) FEBS Lett. 284, 34-38) studies showed that the human E1 alpha gene contains 9 exons, and spans at least 55 kilobases (kb). Exon 1 is 135 bp in length, and contains multiple transcription initiation sites at bases +1, +18, and +22. The complete human E1 alpha cDNA is, therefore, 1,758 bp in length excluding the poly(A)+ tail, and has 27 nucleotides in the 5'-untranslated region. Sequencing of the 5'-flanking region disclosed the absence of a canonical TATA-box in the vicinity of base -30. Several sets of "CAAT" box-like sequences and Sp1 binding-sites are present. Also present are copies of potential AP-2 binding, fat-specific element 1, fat-specific element 2, glucocorticoid-responsive element, and cAMP-responsive element sequences, as well as multiple sets of direct and inverted repeats. The promoter-regulatory region was characterized using deletion constructs and the luciferase reporter assay. The human hepatoma cells (Hep-G2) and Chinese hamster ovary (CHO) cell lines were used as hosts. The results obtained with Hep-G2 cells indicate that the region for high level transcription is located between bases -320 and -115. Extension of the 5'-end of the insert to beyond base -320 markedly reduces promoter activity. The results strongly suggest the presence of inhibitory elements in the region upstream of base -320. Assays in CHO cells show that the region for high level transcription lies between bases -909 and -115. The variation in the region for high level transcription in Hep-G2 and CHO cells may represent cell-type specific differences in the E1 alpha gene promoter function.

Transcriptional regulation of inhibin beta B messenger ribonucleic acid levels in TM.4 or primary rat Sertoli cells by 8-bromo-cyclic adenosine monophosphate.

FSH, a major regulator of inhibin production in the testis, is believed to exert its effects via cAMP second messenger system. Inhibin alpha-subunit gene appears to be regulated by cAMP and has a palindromic cAMP response element sequence TGACGTCA. However, the regulation of the inhibin beta B-subunit gene by cAMP has been less clear. It has been assumed that beta B may not be regulated by cAMP, based mainly on observations that FSH stimulates only alpha, not beta B, mRNA levels, and that the 5'-up-stream regulatory region of the beta B gene does not contain the classical cAMP response element. However, we have observed that 8-bromo-cAMP stimulates beta B mRNA levels in both primary Sertoli (approximately 2-fold) and TM.4 cells (approximately 5-fold). We examined whether this cAMP-induced increase in beta B mRNA levels is the result of increased transcription or altered mRNA stability. Data from nuclear run-on assays demonstrate about a 2-fold increase in relative mRNA synthesis rates in primary Sertoli-cells and about a 4- to 5-fold increase in TM.4 cells. Transfection studies in TM.4 and JEG.3 cell lines with beta B:luciferase chimeric reporter gene constructs containing 1.5 kilobases of the beta B 5'-up-stream regulatory region revealed marked cAMP induction of reporter gene activity in both cell types.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple cis-acting domains mediate basal and adenosine 3',5'-monophosphate-dependent glucagon gene transcription in a mouse neuroendocrine cell line.

A number of cell and tissue-specific differences have been described in studies of the regulation of glucagon gene expression. DNA sequences important for islet cell-specific transcription are not sufficient for expression of the glucagon gene in the intestine, and the posttranslational processing of proglucagon results in the liberation of different peptides in pancreas and intestine. We have studied the control of glucagon gene expression in STC-1 cells, a mouse intestinal neuroendocrine cell line. STC-1 cells are plurihormonal and contain glucagon, somatostatin, amylin, and cholecystokinin, but not insulin mRNA transcripts. Glucagon gene expression is regulated by a cAMP-dependent pathway in STC-1 cells, with an increase in glucagon mRNA transcripts detected 2 h after forskolin stimulation. The levels of glucagon mRNA transcripts remained elevated for 36-48 h after forskolin stimulation, but cycloheximide inhibited the forskolin induction of glucagon gene expression. Although sequences up-stream of -1300 are necessary for intestine-specific glucagon gene transcription in transgenic mice, glucagon-chloramphenicol acetyltransferase (CAT) plasmids containing less than 1300 basepairs of 5'-flanking sequences were transcriptionally active in STC-1 cells. The transcriptional properties of specific DNA elements important for glucagon gene transcription in islet cells differed in STC-1 cells. Deletion of the islet cell-specific enhancer G3 element resulted in an increase in the transcriptional activity of transfected glucagon-CAT plasmids, suggesting that G3 may function as a negative element in STC-1 cells. Deletion of the cAMP response element sequence from -291 to -298 did not eliminate the forskolin induction of glucagon-CAT activity in STC-1 cells, and forskolin responsiveness was maintained with deletions containing only 60 basepairs of rat glucagon gene 5'-flanking sequences. The results of these experiments define novel functional properties for previously characterized domains within the rat glucagon gene 5'-flanking region, suggesting that mouse STC-1 cells may be a useful cell line for studies of the molecular control of glucagon gene expression.

The fine structure of two DNA dodecamers containing the cAMP responsive element sequence and its inverse: Nuclear magnetic resonance and molecular simulation studies

1H and 31P n.m.r. (nuclear magnetic resonance) spectroscopy have been used in conjunction with molecular simulation to determine the structure of two DNA dodecamers. The first of these, CATGACGTCATG, contains the octameric sequence CRE (cAMP responsive element), while the second is the reversed sequence, GTACTGCAGTAC. Structure determination was based on both NOESY (nuclear Overhauser spectroscopy) derived distances and COSY (correlated spectroscopy) dihedral angle data. Access to the 31P spectra also allowed the ε backbone angles to be determined. Considerable care was taken in deriving structural parameters from the n.m.r. data and an excellent level of agreement is obtained with the simulated conformations. Both dodecamers are found to belong to the B-DNA family; however, there is a striking difference between the CRE sequence and its inverse, the former conformation alone showing a strong structural heterogeneity.

Interaction of the -170 cyclic AMP response element with the adjacent CCAAT box in the human fibronectin gene promoter.

In the fibronectin gene promoter the cAMP response element (CRE) and the CCAAT box are separated by only 20 base pairs (bp), i.e. two turns of double helix. Binding of nuclear proteins to these elements, assessed by DNase I footprinting, differs in the different cell types. While in a variety of cells tested (HeLa, granulosa, brain, and adenocarcinoma) only CRE binding activity is observed, liver extracts show both CRE and CCAAT binding activities. Competitions with CRE oligonucleotides were able to prevent the binding of both liver factors, while competitions with CCAAT oligonucleotides only abolished the binding to the CCAAT box. Consistently, the occupation of the CCAAT box was reduced when the distance between the CRE and CCAAT elements was increased in a series of spacing mutants in which DNA fragments of 20, 28, or 44 bp were inserted, and in a construct where the CRE sequence was deleted. Furthermore, the mutants are less efficient than the wild type as templates for in vitro transcription elicited by liver nuclear extracts. Transcriptional activity decreases with the 20- and 28-bp insertions but is partially recovered with the 44-bp insertion. Partial purification of liver CRE- and CCAAT-binding proteins by high performance liquid chromatography on a Mono Q column and recombination of column fractions showed that a novel 73-kDa CRE-binding protein facilitates the association of the CCAAT-binding protein to the CCAAT site of the fibronectin gene.

Binding Specificity of Cyclic Adenosine 3′,5′-Monophosphate- Responsive Element (CRE)-Binding Proteins and Activating Transcription Factors to Naturally Occurring CRE Sequence Variants

The human glycoprotein hormone alpha gene contains a palindromic cAMP-responsive element (CRE), TGACGTCA, which is critical for both basal and cAMP-stimulated expression. Members of the CRE-binding protein (CREB)/activating transcription factor transcription family as well as members of the Jun/Fos family have been shown to bind to this sequence. In nonprimate species, the alpha gene contains a variant of the CRE sequence TGAtGTCA, which differs by a single base substitution. The functional properties of promoters containing variant CREs were examined in transient expression assays in JEG-3 cells (human placental cells) and GH3 cells (rat pituitary cells). Relative to the palindromic CRE, the basal level of expression was reduced with the variant CRE constructions. However, 8-bromo-cAMP stimulated the expression of constructions containing either the palindromic or the variant CREs. Protein interactions with the palindromic and variant CREs were also examined. By Southwestern blot analyses in which proteins were initially separated on a denaturing sodium dodecyl sulfate-gel, transferred to a membrane, and then probed with radio-labeled DNA, several different proteins were bound by either the palindromic or the variant CRE sequences. However, the binding of the variant CRE to CREB was markedly reduced. In nondenaturing gel shift assays, multiple protein-DNA complexes were also formed with both types of CREs, but the variant CRE lacked a major complex that may correspond to homodimers of CREB. To determine the protein composition of these complexes, the gel shift bands were eluted and subjected to Southwestern blot analysis. CREB was identified as one of the protein components in several of the gel shift complexes formed with the variant CRE. We conclude that although the variant CRE binds CREB homodimers poorly, CREB can still interact with this element when associated with other proteins. Thus, variants of the palindromic CRE sequence may provide a mechanism for specifying interactions with different combinations of CREBs.

Molecular characterization of transcription factors that bind to the cAMP responsive region of the substance P precursor gene. cDNA cloning of a novel C/EBP-related factor

A cAMP response element (CRE) plays an important role in the cAMP-mediated gene regulation. Several factors that recognize a CRE have been characterized, and it has been shown that they need either covalent modification by protein kinase A or a cofactor such as the adenovirus Ela to function as an activator. In this study we show that the substance P precursor gene expression is regulated by protein kinase A and identify the CRE sequence in its promoter region. We find that a novel factor and ATF2 bind to the region containing the CRE of the substance P precursor gene. The sequence analysis indicates that the novel protein, designated CELF, has a significant homology to C/EBP gene family proteins in the carboxyl-terminal part containing the basic region and the leucine zipper motif. Ubiquitous expression of CELF suggests that this factor is utilized by various genes. Cell-free transcription analyses indicate that CELF is a constitutive transcriptional activator without apparent phosphorylation by protein kinase A. These results demonstrate that multiple factors are responsible for transcriptional control of the substance P precursor gene through the CRE region.

Structure of theDrosophilaGene for the Laminin B2 Chain

We isolated and sequenced a Drosophila genomic DNA sequence that encodes the entire coding region of the laminin B2 chain. The 11,464-bp genomic sequence contains a 2.1-kb of 5'-flanking DNA, ten exons, nine introns, and the 3'-flanking region. The first exon encodes a 5' untranslated region; the ATG translational start codon is in exon 2. The entire translated region is within a 8.3-kb Eco RI fragment. The Drosophila laminin B2 gene differs substantially in size and exon pattern from those of the human B1 and B2 genes. However, as in the case of the human B1 gene, the overall exon pattern of the Drosophila B2 gene does not correlate well with the highly conserved structural domains and internal repeats of the B2 polypeptide chain. Unlike the human and mouse B1 and B2 genes, the 2.1-kb 5'-flanking region of the Drosophila B2 gene contains a TATA box and two CAAT boxes. Other potential transcriptional regulatory sequences include two reverse complementary cAMP response element sequences; two sequences that are homologous to the retinoic acid response element motifs of the mouse B1 gene; and sequences homologous to the binding sites for transcription factors dFRA and dJRA, zeste, and possibly GAGA. When transfected into Drosophila SL-2 cells, pCAT plasmid containing 2,090 bp of 5'-flanking region shows a 3.0- to 3.5-fold increase in chloramphenicol acetyltransferase activity after induction with retinoic acid and/or 8-bromo-cAMP. These results suggest that this 5'-flanking promoter region may contain DNA sequences that can regulate the expression of the laminin B2 gene.

Novel cyclic adenosine 3',5'-monophosphate response element in the human chorionic gonadotropin beta-subunit gene.

CG is encoded by separate alpha- and beta-subunit genes. Expression of both genes is stimulated by cAMP, but the kinetics of activation are different, with cAMP stimulation of the alpha gene preceding that of the beta gene. The cAMP response element (CRE) in the alpha 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 beta gene had been difficult due to low levels of expression in transfected cells. In this study the 5'-flanking sequence of the CG beta gene was fused to a sensitive luciferase (LUC) reporter gene, allowing delineation of a CG beta CRE in transient expression assays performed in JEG-3 choriocarcinoma cells. The full-length CG beta promoter, -3700 to 362 basepairs (bp), was stimulated 8- to 14-fold by treatment with 1 mM 8-bromo-cAMP. Analyses of a series of deletion mutants in the CG beta promoter demonstrated that -311 CG beta LUC retained nearly complete cAMP stimulation, but deletion to -187 bp eliminated cAMP responsiveness. Overlapping DNA fragments between -311 and -30 bp were fused to a heterologous promoter (-99 alpha LUC) to further define the locations of basal elements and CREs. Basal expression required a combination of at least two distinct elements between -311 and -30 bp, whereas cAMP responsiveness was conferred by sequences between -311 and -202 bp. Shorter DNA sequences within this region were insufficient for cAMP stimulation, suggesting that more than one element may be required. DNase-I footprinting and gel mobility shift studies demonstrated at least three distinct protein-binding sites within the CG beta CRE sequence. Recombinant CREB (expressed in E. coli) did not bind to these sites, and they share no sequence homology with the alpha gene CRE, indicating that a cAMP-responsive transcription factor other than CREB interacts with the CG beta promoter.

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.

Injection of the cAMP-responsive element into the nucleus of Aplysia sensory neurons blocks long-term facilitation

In both vertebrates and invertebrates, long-term memory differs from short-term in requiring protein synthesis during training. Studies of the gill and siphon withdrawal reflex in Aplysia indicate that similar requirements can be demonstrated at the level of sensory and motor neurons which may participate in memory storage. A single application of serotonin, a transmitter that mediates sensitization, to individual sensory and motor cells in dissociated cell cultures leads to enhanced transmitter release from the sensory neurons that is independent of new macromolecular synthesis. Five applications of serotonin cause a long-term enhancement, lasting one or more days, which requires translation and transcription. Prolonged application or intracellular injection into the sensory neuron of cyclic AMP, a second messenger for the action of serotonin, also produce long-term increases in synaptic strength, suggesting that some of the gene products important for long-term facilitation are cAMP-inducible. In eukaryotic cells, most cAMP-inducible genes so far studied are activated by the cAMP-dependent protein kinase (A kinase), which phosphorylates transcription factors that bind the cAMP-responsive element TGACGTCA. The cAMP-responsive element (CRE) binds a protein dimer of relative molecular mass 43,000, the CRE-binding protein (CREBP), which has been purified and shown to increase transcription when phosphorylated by the A kinase. Here we show that extracts of the Aplysia central nervous system and extracts of sensory neurons contain a set of proteins, including one with properties similar to mammalian CREBPs, that specifically bind the mammalian CRE sequence. Microinjection of the CRE sequence into the nucleus of a sensory neuron selectively blocks the serotonin-induced long-term increase in synaptic strength, without affecting short-term facilitation. Taken together, these observations suggest that one or more CREB-like transcriptional activators are required for long-term facilitation.

The specific binding of nuclear protein(s) to the cAMP responsive element (CRE) sequence (TGACGTCA) is reduced by the misincorporation of U and increased by the deamination of C

Point mutations in the cAMP-responsive element (CRE) of the rat somatostatin gene promoter/enhancer sequence (TGACGTCA) were used as a model for assessing the effect of uracil, deriving either from misincorporation during DNA synthesis (T----U) or cytosine deamination (C----U), on the binding of sequence/specific regulatory proteins. The results show that the T----U conversion in both strands of the CRE palindromic sequence reduces its affinity for the CRE binding factor(s), suggesting the crucial role of the methyl group contributed by T for the correct recognition of the sequence. On the other hand, deamination of C in the CpG central dinucleotide (CpG----UpG) causes an increase of binding affinity which is further enhanced by the contemporary deamination in both strands. Then, both uracil misincorporation and cytosine deamination alter the binding to CRE sequence in vitro, suggesting that uracil, if not removed by uracil DNA-glycosylase, could be dangerous for cellular functions.

Identification of Rat Ovarian Nuclear Factors That Interact with the cAMP-inducible Lactate Dehydrogenase A Subunit Promoter

Utilizing the gel electrophoresis/DNA binding assay and a new technique of direct binding of radioactive DNA to protein blots, we have investigated putative factors selective for the cAMP-responsive element (CRE) of the lactate dehydrogenase A subunit promoter in rat ovary nuclear extracts. Analysis of linker-scanning mutants of lactate dehydrogenase A subunit promoter fragments by DNA binding assay identified DNA binding activity selective for the 11-nucleotide sequence 5' TCTGACGTCAG 3' located between positions -51 and -41 relative to the transcription initiation site. This sequence contains the previously identified CRE 5' TGACGTCA 3'. Probing of protein blots with radioactive promoter fragments containing the CRE demonstrated that ovarian nuclear extracts contain a protein of relative molecular mass 47,000 (Mr 47,000) which exhibits selective binding affinity for the CRE. The 47-kDa CRE binding protein was found to be present in comparable levels in the ovaries of normal and hypophysectomized rats. Furthermore, our data suggest the presence of a 37,000-dalton (Mr 37,000) protein which possesses selective binding affinity for part of the CRE sequence. The binding activity/level of the 37-kDa CRE binding protein appeared to be modulated by short-term hypophysectomy/follicle-stimulating hormone administration. These results provide evidence for the presence of CRE binding factors in rat ovarian nuclei, which may be involved in the molecular events responsible for transcriptional regulation of ovarian cAMP-inducible genes.