Putative Cyclic AMP Response Element Research Articles

Identification of promoter response elements that mediate propionate induction of bovine cytosolic phosphoenolpyruvate carboxykinase (PCK1) gene transcription

Cytosolic phosphoenolpyruvate carboxykinase (PCK1) is a key enzyme for gluconeogenesis that is positively regulated by propionate in bovines at the transcription level. The specific elements that determine propionate responsiveness within the bovine PCK1 promoter are unknown. In silico promoter analysis of the bovine PCK1 gene revealed several clusters of transcription factor binding sites. In the present study, we determined the essentiality of the putative cyclic AMP response element (CRE) at -94 through -87 bp and the 2 putative hepatic nuclear factor 4α (HNF4α) binding elements at +68 through +72 and -1,078 through -1,074, respectively, in mediating bovine PCK1 promoter responses to propionate and other regulators, including butyrate, cyclic AMP (cAMP), and glucocorticoids. The wild-type bovine PCK1 promoter [PCK1(WT)] was ligated to a luciferase reporter gene and transfected into rat hepatoma (H4IIE) cells. Activities of PCK1(WT) were induced by approximately 2-, 2-, 4-, 8-, 9-, 18-, and 16-fold respectively when exposed to cAMP (as 1.0 mM 8-Br-cAMP), 5.0 μM dexamethasone, cAMP + dexamethasone, 2.5 mM propionate, cAMP + propionate, cAMP + dexamethasone + propionate, and 2.5 mM butyrate. Seven mutants lacking either one single site, 2 of the 3 sites, or all 3 sites, generated by site-directed mutagenesis, were tested. Responses to propionate and all other treatments were completely abolished when CRE at -94 through -87 bp and HNF4α at +68 through +72 bp were both deleted. Our data indicate that these 2 regulatory elements act synergistically to mediate the bovine PCK1 promoter responses to propionate as well as butyrate, cAMP, and dexamethasone. The activation of PCK1 through these regulatory elements serves to activate the metabolic potential of bovine toward gluconeogenesis when the primary substrate for gluconeogenesis, propionate, is also present.

Involvement of Cyclic AMP Response Element (CRE) in Exendin‐4 Induction of Rat Egr‐1 Gene

We recently observed that exendin-4 (EX-4), a potent agonist of GLP-1, induced cyclin D1 expression through the activation of putative cAMP-responsive element (CRE) site and Egr-1 binding site on cyclin D1 promoter (J Endocrinol, 2006; Diabetologia, 2006, both in press). In the present study, induction of Egr-1 expression by EX-4 and the cis-acting elements involved in the transcriptional activation of Egr-1 gene were investigated in INS-cells. EX-4 (10 nM) rapidly and transiently induced the expressions of Egr-1 mRNA and protein. These inductions were completely inhibited by a specific PKA inhibitor, H-89, suggesting the involvement of cAMP/PKA signaling pathway. In promoter assay using serial-deletion constructs of rat Egr-1 promoter, EX-4 induced the highest promoter activity with full-length constructs (−462 to +27) and the activity of similar degree with the construct deleted up to −124. The construct deleted to −73, which was without serum response elements (SREs) site known to be involved in regulating Egr-1 transcription, still responded to EX-4 moderately. However, further deletion to −46 failed to respond to EX −4. Thus, in examining the role of the nucleotide region from −73 to −46, mutation of the putative CRE site located at −69 resulted in 52% reduction in promoter activity in response to EX-4. This study indicates that the putative CRE site as well as the SRE site is required for the induction of Egr-1 gene by EX-4. This research was grant-supported by Korea Science and Engineering Foundation (R-2004-000-10127-0), Republic of Korea.

Characterization of the prothrombin gene expression during nerve differentiation

The transcriptional regulation of the prothrombin gene expression in neuroblastomas was investigated because of the interest in non-hepatic thrombin expression and function in the nervous system. The data indicated that the murine prothrombin gene was distinctively transcribed in proliferating murine N2a cells and that the transcripts were decreased during the differentiation of N2a cells. The gene transcription in proliferating N2a cells was due to the C-I nuclear complex formation in the promoter region, −248/−140. Mutation analyses indicated that nucleotides from −237 to −231 are the core C-I binding site while the longer sequence −248/−140 is needed for the C-I binding. The C-I binding to the promoter −248/−140 could be inhibited by the presence of competitor probe −187/−166, and the mutation in nucleotides from −186 to −179 significantly diminished not only the formation of C-I binding in the promoter region but also the promoter activity in proliferating neuroblastoma cells. Cyclic AMP response element (CRE) modulator, CREM, appeared to selectively bind to the sequence encompassing −186/−179. Taken together, the results indicate that the prothrombin gene transcription in proliferating N2a cells was critically dependent on the cooperative interaction between the factor(s) binding to the C-1 cis-acting element (−237/−231) and the putative CRE site (−186/−179) in the prothrombin promoter, and that the lack of prothrombin expression that coincided with nerve differentiation was mainly due to the lack of C-I complex formation in the promoter.

Sequencing of the putative promoter region of the cocaine- and amphetamine-regulated-transcript gene and identification of polymorphic sites associated with obesity.

We tested the hypothesis that polymorphisms in the cocaine- and amphetamine-regulated-transcript (CART) gene is associated with the development of obesity. Five-hundred and twenty-eight subjects (325 men and 203 women) aged 49.6+/-11.0 y with body mass index (BMI) of 26.9+/-5.1. The 5(')-flanking region of the CART gene was cloned using adaptor-ligated genomic DNA fragments. The CART gene including the 5(')-flanking region was screened for mutation by PCR-single strand conformation polymorphism and direct sequencing. Associations between polymorphisms and obesity were investigated by PCR-restriction fragment length polymorphism analysis and direct sequencing. The 5(')-flanking region of the CART gene up to -1072 bp from the transcription initiation site was sequenced. The region contained a putative cyclic AMP-responsive element and four E-box motifs upstream of a TATA box. Six polymorphic sites were identified in the upstream region; A-->G at -156, T-->C at -390, T-->G at -484, G-->T at -915, G-->C at -929 and C-->T at -962. The nucleotide substitution at -156 was significantly associated with greater BMI (P=0.036). The allele frequency of the -156 variant was significantly higher in obese subjects with BMI > or = 30 than in non-obese subject (0.41 vs 0.32, P=0.0076). The -929 variant allele in linkage disequilibrium with the -156 variant was also more common in obese subjects. No mutation was found in the coding regions. A single nucleotide insertion/deletion polymorphism at +1355 in the 3(') untranslated region was not associated with obesity. The 5(')-flanking region of the CART gene was highly polymorphic. The -156 polymorphism or polymorphisms in linkage disequilibrium with the site may be associated with genetic predisposition to obesity.

A Novel 14-Base-Pair Regulatory Element Is Essential for In Vivo Expression of Murine β4-Galactosyltransferase-I in Late Pachytene Spermatocytes and Round Spermatids

During murine spermatogenesis, beginning in late pachytene spermatocytes, the beta4-galactosyltransferase-I (beta4GalT-I) gene is transcribed from a male germ cell-specific start site. We had shown previously that a 796-bp genomic fragment that flanks the germ cell start site and contains two putative CRE (cyclic AMP-responsive element)-like motifs directs correct male germ cell expression of the beta-galactosidase reporter gene in late pachytene spermatocytes and round spermatids of transgenic mice (N. L. Shaper, A. Harduin-Lepers, and J. H. Shaper, J. Biol. Chem. 269:25165-25171, 1994). We now report that in vivo expression of beta4GalT-I in developing male germ cells requires an essential and previously undescribed 14-bp regulatory element (5'-GCCGGTTTCCTAGA-3') that is distinct from the two CRE-like sequences. This cis element is located 16 bp upstream of the germ cell-specific start site and binds a male germ cell protein that we have termed TASS-1 (transcriptional activator in late pachytene spermatocytes and round spermatids 1). The presence of the Ets signature binding motif 5'-GGAA-3' on the bottom strand of the TASS-1 sequence (underlined sequence) suggests that TASS-1 is a novel member of the Ets family of transcription factors. Additional transgenic analyses established that an 87-bp genomic fragment containing the TASS-1 regulatory element was sufficient for correct germ cell-specific expression of the beta-galactosidase reporter gene. Furthermore, when the TASS-1 motif was mutated by transversion, within the context of the original 796-bp fragment, transgene expression was reduced 12- to 35-fold in vivo.

A Cyclic AMP Response Element in the Angiotensin-converting Enzyme Gene and the Transcription Factor CREM Are Required for Transcription of the mRNA for the Testicular Isozyme

The angiotensin-converting enzyme (ACE) gene produces two mRNA species from tissue-specific promoters. The transcription start site of the mRNA for the smaller testicular isozyme (ACET) is located within an intron of the larger transcription unit that encodes the pulmonary isozyme (ACEP).We have previously demonstrated that a 298-base pair DNA fragment, 5' to the rabbit ACET mRNA transcription initiation site, can activate the testicular expression of a transgenic reporter gene. In the current study, using the same transgenic reporter system, we identified a putative cyclic AMP response element present within this DNA fragment to be absolutely essential for transcriptional activation. Moreover, we observed that ACET mRNA was not expressed in the testes of mice homozygous for a null mutation in the transcription factor CREM. However, in the same mice, ACEP mRNA was abundantly expressed in the lung. Our observations indicate that ACET mRNA expression in the testes is regulated by the putative cyclic AMP response element present 5' to the transcription start site and the corresponding transcription factor CREM.

The Cyclic AMP Response Elements of the Genes for Angiotensin-converting Enzyme and Phosphoenolpyruvate Carboxykinase (GTP) Can Mediate Transcriptional Activation by CREMτ and CREMα

The potential of the CREM family of proteins to activate transcription of the genes encoding the testis-specific isozyme of angiotensin converting enzyme (ACET) and the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (GTP) (PEPCK) (EC 4.1.1.32) were investigated. Both CREM tau and CREM alpha bind efficiently to the putative cyclic AMP response element (CRE) present in the ACET gene (CRET) and to the CRE in the PEPCK gene. In HepG2 cells, the CRE was required for the strong stimulation by CREM tau of the expression of a chimeric PEPCK (-210 to +73)-chloramphenicol acetyl transferase (CAT) gene. The CRE could be mutated to the CRET sequence without losing the stimulatory effects of CREM tau. However, a similar chimeric gene driven by the regulatory region of the ACET gene, which contains the CRET site, could only be stimulated by CREM tau when its imperfect TATA element was mutated to an authentic TATA. Surprisingly, CREM alpha, an alleged inhibitor of CRE-mediated transcription, stimulated the expression of both PEPCK-CAT and ACET-CAT genes in HepG2 cells, a process which required the presence of the CRE and the CRET sites, respectively. In contrast, when the same CRE elements were used to drive the transcription of a chimeric gene containing the thymidine kinase promoter linked to the CAT structural gene, CREM alpha inhibited its expression in HepG2 and JEG3 cells. The expression of the same chimeric gene, however, was stimulated by CREM alpha in F9 embryonal carcinoma cells. These results demonstrated that the nature of the transcriptional effects of CREM isoforms on CRE-mediated transcription depends on the specific gene, the specific cell type and the promoter context of the CRE site.

Isolation and characterization of the human spr1 gene and its regulation of expression by phorbol ester and cyclic AMP

The small proline-rich protein gene (spr1) is a marker whose expression is frequently associated with squamous cell differentiation. We observed that the expression of the spr1 gene is strongly induced by phorbol 12-myristate 13-acetate (PMA). Both the time course result and the nuclear run-on transcriptional assay suggested that the regulation of spr1 expression by PMA is controlled at the transcriptional level. To understand the nature of this regulation, human genomic clones of the spr1 gene were isolated. DNA sequence analysis revealed that the human spr1 gene contains two exons and a single intron located within the 5'-untranslated region. An AP-1 binding site (TGAGTCA) is found at -142, and a putative cyclic AMP-responsive element (TGAGGTCA) at -597 base pairs upstream of the transcription start site. A chimeric construct containing the 5'-flanking region of the spr1 gene and the chloramphenicol acetyltransferase (CAT) reporter gene was used to transfect HeLa cells or monkey primary TBE cells. The CAT activity in transfected cells is stimulated 7.5-11-fold by PMA, and the stimulation is inhibited by a protein kinase C inhibitor or by pretreating cells with PMA to down-regulate the protein kinase C activity. The CAT activity is also stimulated 3.5-fold by dibutyryl cyclic AMP, a protein kinase A activator. The stimulations by PMA and cAMP are additive. These results suggest that protein kinase C and probably protein kinase A play important roles in regulating the transcription of the spr1 gene.

The human I alpha 1 and I alpha 2 germline promoter elements: proximal positive and distal negative elements may regulate the tissue specific expression of C alpha 1 and C alpha 2 germline transcripts.

Treatment of human splenic B lymphocytes with the mitogen Branhamella catarrhalis (BC) and transforming growth factor-beta 1 (TGF-beta 1) induces expression of germline Ig C alpha transcripts and class switching to this isotype. To further characterize the molecular mechanism by which TGF-beta 1 and mitogenic signals regulate the expression of unrearranged C alpha 1 and C alpha 2 genes, we have characterized the promoter elements that are responsible for the transcriptional activation of their corresponding germline genes using transient expression assays. We report here that both in the I alpha 1 and the I alpha 2 regions, maximal phorbol myristate acetate (PMA) and TGF-beta 1 responsiveness of the promoters can be conferred by 327 bp spanning the transcription initiation sites and a previously identified phylogenetically conserved region. The expression of these 327 bp segments is not restricted to the B cell lineage since they are also active in the erythroleukemia cell line K562 as well as the B cell lines Raji and DG75. Mutational analyses have demonstrated the importance of sequences within the 327 bp segment that contain a putative cyclic AMP responsive element binding protein (CREB) binding site for TGF-beta 1 and PMA responsiveness and putative PU-1 and Sp1 binding sites for basal promoter activity. Upstream distal elements that could negatively modulate the expression of the I alpha 1 and I alpha 2 promoters, particularly in non-B cells, have been identified. Three such elements were mapped between positions -352 to -243, -627 to -516, and upstream of position -731 respectively. The influence of these elements presumably contributes to the B cell specific expression of the I alpha 1 and I alpha 2 promoters. The I alpha 1 and I alpha 2 promoters were found to be functionally indistinguishable from each other with respect to their basal level of expression, and their responsiveness to TGF-beta 1.

Sperm-specific expression of angiotensin-converting enzyme (ACE) is mediated by a 91-base-pair promoter containing a CRE-like element.

The gene encoding the testis isozyme of angiotensin-converting enzyme (testis ACE) is one example of the many genes expressed uniquely during spermatogenesis. This protein is expressed by developing germ cells late in their development and results from the activation of a sperm-specific promoter that is located within intron 12 of the gene encoding the somatic isozyme of ACE. In vitro transcription, DNase footprinting, gel shift assays, and transgenic mouse studies have been used to define the minimal testes ACE promoter and to characterize DNA-protein interactions mediating germ cell-specific expression. These studies show that proper cell- and stage-specific expression of testis ACE requires only a small portion of the immediate upstream sequence extending to -91. A critical motif within this core promoter is a cyclic AMP-responsive element sequence that interacts with a testis-specific transactivating factor. Since this putative cyclic AMP-responsive element has been conserved within the testis ACE promoters of different species and is found at the same site in other genes that are expressed specifically in the testis, it may provide a common mechanism for the recognition of sperm-specific promoters.

Genomic organization and expression of the human alpha 1B-adrenergic receptor.

alpha 1-Adrenergic receptors (ARs) are members of the guanine nucleotide-binding protein-coupled receptor superfamily. The genes for all ARs described thus far are intronless. We report here the cloning and the nucleotide sequence of the gene for the human alpha 1B-AR. It consists of two exons and a single large intron of at least 20 kilobases which interrupts the coding region at the end of the putative sixth transmembrane domain. The deduced amino acid sequence of the encoded receptor has a high degree of homology to the cloned hamster, rat, and dog alpha 1B-ARs. To characterize the encoded protein, a fusion gene constructed by splicing together exon 1 and exon 2 was expressed transiently in COS-1 cells. The transfected gene fusion product resulted in the production of an alpha 1B-AR with ligand binding characteristics indistinguishable from those of the expressed hamster alpha 1B cDNA. Evidence that the human alpha 1B-AR gene we have isolated is indeed transcribed is the finding of similar sized (2.8-kilobase) transcripts in human heart and other tissues by Northern blot analysis when either exon 1 or exon 2 is used as a probe. Moreover, using primers designed to span the exon 1/exon 2 boundary, a polymerase chain reaction product generated from single-stranded DNA prepared from human heart mRNA had the exact size and nucleotide sequence predicted for a transcript in which exon 1 is spliced to exon 2. The 5'-flanking region (924 base pairs (bp)) of exon 1 contains neither a TATA box nor a CAAT box but is high in GC content (70%) and contains several Sp1 binding sites (GC boxes), consistent with promoters described for housekeeping genes. The 5'-untranslated region also contains a putative cyclic AMP response element. Primer extension studies and RNase protection assays suggested that there are several potential transcription start sites in most tissues with a predominant site located 173 bp upstream from the translation start site. The 3'-flanking region contains a putative polyadenylation signal (ATTAAA) 492 bp downstream from the stop codon. The genomic organization of the human alpha 1B-AR with a single large intron interrupting its coding region differs from those of other ARs as well as muscarinic and 5-hydroxy-tryptamine receptors, which are intronless. The location of the intron in the human alpha 1B-AR gene is also unique among those members of the G-protein-coupled receptor family that do possess introns.(ABSTRACT TRUNCATED AT 400 WORDS)

Structural differences in 5′-flanking regions of rat cytochrome P-450aldo and P-45011β genes

Two rat genomic clones, one for cytochrome P-450aldo and the other for P-450(11) beta, were isolated and characterized. The two genes, encoding structurally homologous proteins, were closely similar in their intron-exon organizations. Their 5'-flanking regions, however, contained only a few homologous regions. A putative cyclic AMP responsive element, TGACGTGA, was found in the P-450aldo gene, but this sequence was altered at two positions in the P-450(11) beta gene. S1 nuclease protection assay revealed a single transcription initiation site for the P-450aldo gene, while multiple sites were found for the P-450(11) beta gene. These results suggest that transcriptional regulation of the rat P-450aldo and P-450(11) beta genes is due to differences in the sequences of their 5'-flanking regions.

Regulation of mouse ornithine decarboxylase gene expression in a macrophage-like cell line: Synergistic induction by bacterial lipopolysaccharide and camp

The ability of the promotor/enhancer region of the mouse ornithine decarboxylase gene to respond to various stimuli was studied. This region was subcloned into multiple fragments and these were inserted in front of the chloramphenicol acetyltransferase gene on an expression vector, pBLCAT3. These ODC/CAT constructs were transfected into a mouse macrophage-like cell line, RAW264. The transfected cells were stimulated by bacterial lipopolysaccharide, 8-bromo cAMP or both followed by analysis of chloramphenicol acetyltransferase activity. Optimal inducible chloramphenicol acetyltransferase expression was obtained when sequences from -90 to +12 (with respect to the transcriptional start site) were tested in cells treated with a combination of lipopolysaccharide and 8-bromo cAMP. A putative cyclic AMP response element located at -48 was altered by site-directed mutagenesis but these alterations did not diminish activity in response to stimulation with lipopolysaccharide and 8-bromo cAMP.