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PubMed2001

5-Aminolaevulinate synthase gene promoter contains two cAMP-response element (CRE)-like sites that confer positive and negative responsiveness to CRE-binding protein (CREB).

The first and rate-controlling step of the haem biosynthetic pathway in mammals and fungi is catalysed by the mitochondrial-matrix enzyme 5-aminolaevulinate synthase (ALAS). The purpose of this work was to explore the molecular mechanisms involved in the cAMP regulation of rat housekeeping ALAS gene expression. Thus we have examined the ALAS promoter for putative transcription-factor-binding sites that may regulate transcription in a cAMP-dependent protein kinase (PKA)-induced context. Applying both transient transfection assays with a chloramphenicol acetyltransferase reporter gene driven by progressive ALAS promoter deletions in HepG2, and electrophoresis mobility-shift assays we have identified two putative cAMP-response elements (CREs) at positions -38 and -142. Functional analysis showed that both CRE-like sites were necessary for complete PKA induction, but only one for basal expression. Co-transfection with a CRE-binding protein (CREB) expression vector increased PKA-mediated induction of ALAS promoter transcriptional activity. However, in the absence of co-transfected PKA, CREB worked as a specific repressor for ALAS promoter activity. A CREB mutant deficient in a PKA phosphorylation site was unable to induce expression of the ALAS gene but could inhibit non-stimulated promoter activity. Furthermore, a DNA-binding mutant of CREB did not interfere with ALAS promoter basal activity. Site-directed-mutagenesis studies showed that only the nearest element to the transcription start site was able to inhibit the activity of the promoter. Therefore, we conclude that CREB, through its binding to CRE-like sites, mediates the effect of cAMP on ALAS gene expression. Moreover, we propose that CREB could also act as a repressor of ALAS transcription, but is able to reverse its role after PKA activation. Dephosphorylated CREB would interfere in a spatial-disposition-dependent manner with the transcriptional machinery driving inhibition of gene expression.

5-Aminolevulinate Synthetase↗
PubMed1996

Differential binding of cAMP-responsive-element (CRE)-binding protein-1 and activating transcription factor-2 to a CRE-like element in the human tissue-type plasminogen activator (t-PA) gene promoter correlates with opposite regulation of t-PA by phorbol ester in HT-1080 and HeLa cells.

The human tissue-type plasminogen activator gene (t-PA) is induced by the phorbol ester, phorbol 12-myristate 13-acetate (PMA), in HeLa cells. Previous studies in transfected HeLa cells identified two cis-acting regulatory elements within the t-PA gene promoter responsible for both constitutive and PMA-inducible expression. One element differs from the consensus cAMP response element (CRE) by a single nucleotide substitution (referred to in this report as t-PACRE) and another which bears similarity to the AP-2 recognition sequence. In HT-1080 fibrosarcoma cells, t-PA mRNA levels are expressed at higher constitutive levels and are suppressed by PMA. Nuclear run-on transcription experiments indicate that PMA-mediated suppression of t-PA in these cells is associated with a decrease in t-PA gene template activity. We designed experiments to determine whether nuclear t-PACRE or AP-2-like binding proteins were differentially expressed in HeLa and HT-1080 cells and, accordingly, if these could be correlated with the opposite effect of PMA on t-PA expression. Band shift analyses indicated that the migration profiles of HeLa and HT-1080 nuclear proteins interacting with the AP-2-like site were indistinguishable; however, those produced with the t-PACRE binding site were qualitatively and quantitatively distinct. The distribution of t-PACRE binding proteins in these cells was investigated in a supershift assay using specific antibodies against members of the fos/jun and CRE-binding protein (CREB)/activating transcription factor (ATF) families. In HT-1080 cells, CREB-1 was the most prominent t-PACRE-binding activity detected and was greatly increased in cells treated with PMA. In contrast, CREB-1 activity was absent in HeLa cells, but antibodies specific for ATF-2 produced a marked supershifted complex which was unaffected by PMA treatment. Since CREB-1 can repress transcription of other target genes (including c-jun) via association with identical cis-acting CRE-like sequences, we suggest that the mechanism for the transcriptional down-regulation of t-PA by PMA in HT-1080 cells requires CREB-1 binding to the t-PACRE while ATF-2, by associating with the same site, plays a role in PMA-mediated induction of t-PA in HeLa cells.

Activating Transcription Factor 2↗
PubMed2004

Targeted insertion of an IRES Cre into the Hnf4alpha locus: Cre-mediated recombination in the liver, kidney, and gut epithelium.

The Hnf4alpha gene belongs to a family of trancriptional regulators required for liver development and function. Hnf4alpha is also expressed in other tissues, including the newly formed visceral endoderm of the early postimplantation embryo, and later in embryogenesis in the gut epithelium and the kidney. The regulatory sequences involved in controlling expression of Hnf4alpha at these diverse sites are not clearly understood. Here we used homologous recombination to introduce Cre recombinase coding sequences into the endogenous Hnf4alpha locus. Crossing Hnf4alpha(Creex2/+) mice with R26R partners allowed us to follow the pattern of Cre-mediated recombination. Our results show that recombination of the reporter allele closely follows endogenous Hnf4alpha expression, but with a slight temporal delay. Thus, the Hnf4alpha(Creex2) strain should prove useful for conditionally deleting gene activity in the liver, gut epithelium, or kidney.

Animals↗
PubMed2004

N-methyl-D-aspartate induces phosphorylation of cAMP response element (CRE)-binding protein and increases DNA-binding activity of CRE in rat retina.

The aim of this study was to investigate whether an excitotoxic concentration of N-methyl-D-aspartate (NMDA) increases the expression of the phosphorylated cAMP response element-binding protein (p-CREB) and the DNA-binding activity of the cAMP response element (CRE) in rat retina. Intravitreal injection of NMDA was performed in adult male Wistar rats. p-CREB protein levels in the retina were examined by Western blot analysis. DNA-binding activity of CRE in the retina was evaluated by an electrophoretic mobility gel shift assay (EMSA). We confirmed that NMDA induced the reduction of ganglion cells and the inner plexiform layer of the retina. Western blot analysis showed increases in the expression of p-CREB in the retina 12 and 24 h after intravitreal NMDA injection and dimer formation of CREB in the nuclear fraction at 24 h. Increases of DNA-binding activity were observed in the retina 24 h after NMDA injection by EMSA. Our results suggest that phosphorylation of CREB may involved in NMDA-induced excitotoxicity in rat retina. Phosphorylated CREB seems to be the active form and the one that is transcribed.

Animals↗
PubMed2001

Characterization of a novel EGFP reporter mouse to monitor Cre recombination as demonstrated by a Tie2 Cre mouse line.

The use of the Cre/loxP system has greatly empowered the field of gene targeting. Here we describe the successful establishment of a novel knock-in EGFP reporter mouse line to monitor Cre-induced recombination in the vast majority of cell types. The value of this reporter mouse line is demonstrated by the use of a novel Tie2Cre transgenic mouse line that facilitates gene targeting in endothelial and hematopoietic cells. High efficiency of recombination was found in all endothelial cells and in the majority of hematopoietic cells but was absent in other tissues. Furthermore, in the second generation, the Tie2Cre mouse can be used to get 100% recombination of one allele, whilst allowing tissue specific in the second, therefore offering excellent efficiency.

Alleles↗
PubMed2001

Impaired adipogenesis and lipolysis in the mouse upon selective ablation of the retinoid X receptor alpha mediated by a tamoxifen-inducible chimeric Cre recombinase (Cre-ERT2) in adipocytes.

Retinoid X receptor alpha (RXRalpha) is involved in multiple signaling pathways, as a heterodimeric partner of several nuclear receptors. To investigate its function in energy homeostasis, we have selectively ablated the RXRalpha gene in adipocytes of 4-week-old transgenic mice by using the tamoxifen-inducible Cre-ERT2 recombination system. Mice lacking RXRalpha in adipocytes were resistant to dietary and chemically induced obesity and impaired in fasting-induced lipolysis. Our results also indicate that RXRalpha is involved in adipocyte differentiation. Thus, our data demonstrate the feasibility of adipocyte-selective temporally controlled gene engineering and reveal a central role of RXRalpha in adipogenesis, probably as a heterodimeric partner for peroxisome proliferator-activated receptor gamma.

Adipocytes↗
PubMed1995

Nuclear localization of v-Abl leads to complex formation with cyclic AMP response element (CRE)-binding protein and transactivation through CRE motifs.

Deregulated expression of v-abl and BCR/abl genes has been associated with myeloproliferative syndromes and myelodysplasia, both of which can progress to acute leukemia. These studies identify the localization of the oncogenic form of the abl gene product encoded by the Abelson murine leukemia virus in the nuclei of myeloid cells and the association of the v-Abl protein with the transcriptional regulator cyclic AMP response element-binding protein (CREB). We have mapped the specific domains within each of the proteins responsible for this interaction. We have shown that complex formation is a prerequisite for transcriptional potentiation of CREB. Transient overexpression of the homologous cellular protein c-Abl also results in the activation of promoters containing an intact CRE. These observations identify a novel function for v-Abl, that of a transcriptional activator that physically interacts with a transcription factor.

Base Sequence↗
PubMed1993

Regulation of trans-activating capacity of CRE-BPa by phorbol ester tumor promoter TPA.

CRE-BPa, here designated as CRE-BPa alpha, is a novel member of the CRE (cAMP response element)-binding protein CRE-BP1 family. CRE-BPa alpha has four regions highly homologous to CRE-BP1, including a putative metal finger structure and a DNA-binding domain consisting of a basic amino acid cluster and a leucine zipper. CRE-BPa specifically binds to CRE as a homodimer or heterodimer with c-Jun or CRE-BP1. Here we report three alternative splicing forms of CRE-BPa alpha: two of them, CRE-BPa beta and CRE-BPa gamma, lack the N-terminal 7 and 33 amino acids of CRE-BPa alpha, and the third one CRE-BPa delta, has 16 additional amino acids in the N-terminus and amino acids 156-508 of CRE-BPa alpha. In CAT cotransfection experiments using CV-1 cells, transient expression of each of four CRE-BPa proteins caused a 1.6- to 3.4-fold increase of CRE-dependent transcription, respectively. Interestingly, these weak trans-activating capacities of CRE-BPa proteins were enhanced 2.7- to 3.6-fold by treatment of cells with 12-O-tetradecanoyl-phorbol 13-acetate (TPA). However, CRE-BPa did not affect the TPA-induced and TRE (TPA response element)-dependent transcription. These results indicate that CRE-BPa is a CRE-dependent trans-activator, and that CRE-BPa can confer TPA inducibility on CRE. Thus, CRE-BPa has an unique characteristic of cross-talk between cAMP pathway and TPA pathway.

Activating Transcription Factor 2↗
PubMed1993

Isolation and characterization of a novel member of the gene family encoding the cAMP response element-binding protein CRE-BP1.

Among multiple CRE (cyclic AMP response element)-binding proteins, CRE-BP1 (also designated ATF-2) has two unique characteristics: it mediates the adenovirus E1A-induced trans-activation and forms a heterodimer with c-Jun. Two structures, a putative metal finger and a leucine zipper, in CRE-BP1 are responsible for these capacities. As a new member of a CRE-BP1 family that has similar metal finger and leucine zipper structures, we have isolated cDNA clones of CRE-BPa by cross-hybridization with CRE-BP1 cDNA. CRE-BPa protein consists of 508 amino acids and has a molecular weight of 56,840. CRE-BPa protein is highly homologous with CRE-BP1 in four regions: two of them are the regions containing the putative metal finger or the DNA-binding domain consisting of the basic amino acid cluster and the leucine zipper. Like CRE-BP1, CRE-BPa binds to CRE with higher affinity than to the 12-O-tetradecanoylphorbol-13-acetate response element as a homodimer or a CRE-BPa/c-Jun or CRE-BPa/CRE-BP1 heterodimer. However, using the c-Myb-CRE-BPa fusion protein, it was show that CRE-BPa could not mediate the E1A-induced trans-activation. Expression of CRE-BPa mRNA was found in a limited number of cell lines, and multiple sizes of CRE-BPa mRNA species were detected in some cell lines and tissues. CRE-BPa will be useful to clarify the mechanism of CRE-mediated transcriptional activation by E1A or c-Jun.

Activating Transcription Factor 2↗
PubMed2005

Practical range of effective dose for Cre recombinase-expressing recombinant adenovirus without cell toxicity in mammalian cells.

The site-specific recombinase Cre is valuable for regulation of gene expression not only in vitro but also in vivo. We previously reported that replication-deficient recombinant adenovirus (rAd) expressing Cre can mediate efficient and strict regulation in 100% of cultured cells. Recently, the constitutive-expression of Cre using retrovirus or lentivirus vector reportedly inhibited cell-growth, but the effect of transient Cre expression have not yet been examined. Here we showed that an excess amount of Cre produced from Cre-expressing rAd caused a deleterious effect in cells even when Cre was transiently expressed. We used three rAds carrying promoters with different activities: the SV40 early promoter (AxSVENCre), the SR alpha promoter (AxSRCre) and the CAG promoter (AxCANCre). Cell toxicity was clearly caused by Cre itself and was distinguishable from that caused by rAd virions when the cytopathic effects of these rAds were compared with that of a control virus lacking the Cre expression unit. Cre toxicity was strongly correlated with the expression level of Cre. Importantly, AxSRCre and AxCANCre gave a 60-fold range of effective MOIs ("effective range") sufficient for gene activation without causing cell toxicity from either the rAd particles or Cre itself, while AxSVENCre failed to give such a range because the expression level of Cre was too low. When Cre was tagged with a nuclear localization signal (NLS), not only its activity but also Cre toxicity was increased fourfold, and the effective range was unchanged. Therefore, AxSRNCre might be more useful to control cell toxicity from the rAd virions than AxSRCre. Cre-induced cell toxicity can be avoided by pre-examining the "effective range" using the purpose cell lines before starting experiments utilizing the experiment of Cre-expressing rAd.

Adenoviridae↗
PubMed1986

Bacteriophage P1 cre gene and its regulatory region. Evidence for multiple promoters and for regulation by DNA methylation.

The bacteriophage P1 site-specific recombination system consists of two components, a site, loxP, at which recombination occurs, and a recombinase protein, Cre. In this paper, we present the DNA sequence of the cre structural gene and its upstream regulatory region. Analysis of the sequence indicates: (1) that cre encodes a protein of 343 amino acids; (2) that cre and loxP are separated by a 434 base-pair region that contains a 73 amino acid open reading frame, orf1; and (3) that cre and orf1 are oriented with their amino-terminal ends proximal to loxP. We have identified three promoters that are located upstream of the cre structural gene. Their activities range from 7 to 10% of the activity of the galactose operon promoter. The promoter furthest from cre, pR1, contains two Dam methylation sites (5'-G-A-T-C-3') in its -35 region, and is sensitive to Dam methylation. Its transcription is three- to fourfold higher in a dam- host than it is in a dam+ host. The promoter closest to cre, pR3, signals the production of an RNA transcript that functions inefficiently for Cre protein synthesis because it lacks a ribosome recognition site. None of the three cre promoters is sensitive to proteins expressed by the P1 prophage, including the c1 repressor protein. To assess the role of cre in the P1 life-cycle, we isolated cre mutants and studied their behavior in recA+ and recA- hosts. Those studies indicate that Cre is dispensable for viral vegetative growth and lysogeny in a recA+ host, but is required for both processes in a recA- host. The cre requirement for lysogeny suggests that the protein is essential for the cyclization of newly injected terminally redundant virion DNA. The requirement for vegetative growth suggests that Cre also has a role to play in the viral lytic cycle after the viral DNA has been cyclized.

Base Sequence↗
PubMed2004

Expression and activity of osteoblast-targeted Cre recombinase transgenes in murine skeletal tissues.

The Cre/loxP recombination system can be used to circumvent many of the limitations of generalized gene ablation in mice. Here we present the development and characterization of transgenic mice in which Cre recombinase has been targeted to cells of the osteoblast lineage with 2.3 kb (Col 2.3-Cre) and 3.6 kb (Col 3.6-Cre) fragments of the rat Col1a1 promoter. Cre mRNA was detected in calvaria and long bone of adult Col 2.3-Cre and Col 3.6-Cre mice, as well as in tendon and skin of Col 3.6-Cre mice. To obtain a historical marking of the temporal and spatial pattern of Cre-mediated gene rearrangement, Col-Cre mice were bred with ROSA26 (R26R) mice in which Cre-mediated excision of a floxed cassette results in LacZ expression. In Col 2.3-Cre;R26R and Col 3.6-Cre;R26R progeny, calvarial and long bone osteoblasts showed intense beta-gal staining at embryonic day 18 and postnatal day 5. The spatial pattern of beta-gal staining was more restricted in bone and in bone marrow stromal cultures established from Col 2.3-Cre;R26R mice. Similar differences in the spatial patterns of expression were seen in transgenic bone carrying Col1a1-GFP visual reporters. Our data suggest that Col 2.3-Cre and Col 3.6-Cre transgenic mice may be useful for conditional gene targeting in vivo or for obtaining osteoblast populations for in vitro culture in which a gene of interest has been inactivated.

Animals↗
PubMed2004

Efficient delivery of Cre-recombinase to neurons in vivo and stable transduction of neurons using adeno-associated and lentiviral vectors.

BACKGROUND: Inactivating genes in vivo is an important technique for establishing their function in the adult nervous system. Unfortunately, conventional knockout mice may suffer from several limitations including embryonic or perinatal lethality and the compensatory regulation of other genes. One approach to producing conditional activation or inactivation of genes involves the use of Cre recombinase to remove loxP-flanked segments of DNA. We have studied the effects of delivering Cre to the hippocampus and neocortex of adult mice by injecting replication-deficient adeno-associated virus (AAV) and lentiviral (LV) vectors into discrete regions of the forebrain. RESULTS: Recombinant AAV-Cre, AAV-GFP (green fluorescent protein) and LV-Cre-EGFP (enhanced GFP) were made with the transgene controlled by the cytomegalovirus promoter. Infecting 293T cells in vitro with AAV-Cre and LV-Cre-EGFP resulted in transduction of most cells as shown by GFP fluorescence and Cre immunoreactivity. Injections of submicrolitre quantities of LV-Cre-EGFP and mixtures of AAV-Cre with AAV-GFP into the neocortex and hippocampus of adult Rosa26 reporter mice resulted in strong Cre and GFP expression in the dentate gyrus and moderate to strong labelling in specific regions of the hippocampus and in the neocortex, mainly in neurons. The pattern of expression of Cre and GFP obtained with AAV and LV vectors was very similar. X-gal staining showed that Cre-mediated recombination had occurred in neurons in the same regions of the brain, starting at 3 days post-injection. No obvious toxic effects of Cre expression were detected even after four weeks post-injection. CONCLUSION: AAV and LV vectors are capable of delivering Cre to neurons in discrete regions of the adult mouse brain and producing recombination.

Animals↗