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PubMed1998

Novel CRE-binding proteins of 11-16 kDa bind to the LDH A-gene CRE in a sequence specific and hepatocyte-growth dependent manner in partially hepatectomized rat liver.

We examined cAMP response element (CRE)-binding proteins involved in lactate dehydrogenase A (LDHA)-gene transcription in rat liver after partial hepatectomy. Gel retardation and Southwestern blot assays showed that the CRE-binding activity of the 11-16 kDa novel proteins increased in accordance with increases in LDH A-mRNA in regenerating liver tissues, whereas that of the 43 kDa CREB did not. Using CRE-oligonucleotide affinity chromatography and reverse-phase HPLC, we purified four CRE-binding proteins of 11.2, 15.2, 15.8, and 16.3 kDa. N-terminal amino acid sequences of 15.2 and 16.3 kDa proteins revealed a high sequence homology to but were not identical with those of rat histone H2A.1 and H2B, respectively. CRE-bindings of these two proteins were highly specific, while those of histones H2A.1 and H2B were nonspecific as shown by competition-Southwestern blot and DNase I footprinting assays. Taking these data together, we suggest that the novel 11-16 kDa CRE-binding proteins are responsible for the cell growth-dependent inducibility of LDH A-gene transcription during liver regeneration.

Amino Acid Sequence↗

PubMed1999

Dual blockade of cyclic AMP response element- (CRE) and AP-1-directed transcription by CRE-transcription factor decoy oligonucleotide. gene-specific inhibition of tumor growth.

Alteration of gene transcription by inhibition of specific transcriptional regulatory proteins has important therapeutic potential. Synthetic double-stranded phosphorothioate oligonucleotides with high affinity for a target transcription factor can be introduced into cells as decoy cis-elements to bind the factors and alter gene expression. The CRE (cyclic AMP response element)-transcription factor complex is a pleiotropic activator that participates in the induction of a wide variety of cellular and viral genes. Because the CRE cis-element, TGACGTCA, is palindromic, a synthetic single-stranded oligonucleotide composed of the CRE sequence self-hybridizes to form a duplex/hairpin. Herein we report that the CRE-palindromic oligonucleotide can penetrate into cells, compete with CRE enhancers for binding transcription factors, and specifically interfere with CRE- and AP-1-directed transcription in vivo. These oligonucleotides restrained tumor cell proliferation, without affecting the growth of noncancerous cells. This decoy oligonucleotide approach offers great promise as a tool for defining cellular regulatory processes and treating cancer and other diseases.

Blotting, Western↗

PubMed2001

New variants of inducible Cre recombinase: a novel mutant of Cre-PR fusion protein exhibits enhanced sensitivity and an expanded range of inducibility.

We have developed a novel inducible Cre mutant with enhanced recombinase activity to mediate genetic switching events. The protein, designated Cre*PR, is composed of a new Cre mutant at the N-terminus followed by the ligand-binding domain (LBD) of the progesterone receptor (PR). The response to low doses of inducer is significantly enhanced by elongating the C-terminus of the PR LBD from amino acid 891 to 914. The mutant Cre lacks the first 18 amino acids and contains a Val-->Ala substitution at position 336, thereby destroying a cryptic splice donor at the 3'-end of CRE: The latter mutation reduces unwanted background recombinase activity in the absence of the synthetic ligand RU486 by a factor of at least 10 to an almost undetectable level. Thus, the recombinase activity turns out to be inducible by a factor of >200. We expect Cre*PR to serve as a valuable tool for conditional expression of genes both in vitro and in vivo.

Amino Acid Substitution↗

PubMed2002

Ethanol stimulates cAMP-responsive element (CRE)-mediated transcription via CRE-binding protein and cAMP-dependent protein kinase.

Alcoholism is characterized by tolerance, dependence, and unrestrained craving for alcohol. Adaptive responses, including changes in gene expression in neurons, are thought to account for some of these complex behavioral abnormalities. We have shown in the NG108-15 neuroblastoma x glioma hybrid cell line that ethanol increases cellular cAMP levels via activation of adenosine A(2) receptors, leading to phosphorylation of the cAMP response element-binding protein (CREB). However, phosphorylation of CREB is not sufficient to activate cAMP response element (CRE)-mediated gene expression. Here we investigate whether ethanol increases CRE-mediated gene expression via endogenous CREB using a CRE-regulated luciferase reporter construct, transfected into NG108-15 cells. We find increased luciferase activity as a function of time of exposure to ethanol. Coexpression of a dominant-negative CREB construct blocked ethanol-stimulated CRE-luciferase expression, further suggesting that CREB is required for this response. We also determined whether ethanol-induced increases in gene expression are mediated by ethanol-induced increases in extracellular adenosine. We found that CRE-mediated gene expression induced by ethanol occurs in two phases: an early phase (4 h), in which adenosine receptor blockade prevents ethanol-induced gene expression, and a later phase (14 h), which is not blocked by an adenosine receptor antagonist. In both phases, inhibition of cAMP-dependent protein kinase A (PKA) activity prevented ethanol-induced CRE-mediated luciferase expression. Our data suggest that ethanol induces cAMP-dependent gene expression regulated by CREB and PKA and that this signaling pathway may mediate some of the addictive behaviors underlying alcoholism.

Animals↗

PubMed2001

Cre stoplight: a red/green fluorescent reporter of Cre recombinase expression in living cells.

The Cre/lox system is a powerful genetic tool with which to manipulate the genome. Here, we describe the development of a simple reporter system for Cre recombinase, called the Cre Stoplight. In the absence of Cre, the red fluorescent protein is expressed; when Cre catalyzes a recombination event, the green fluorescent protein is produced. Testing this system in transiently transfected cells showed that it produced robust signals (90% of the cells converted from red to green) when equal amounts of the plasmids encoding Cre recombinase and the Cre Stoplight were used. A 1:100 ratio of enzyme to reporter plasmid produced similar results, and a 1:10000 ratio was necessary to significantly reduce the number of cells converting to green (1%).

Cell Line↗

PubMed2002

Efficient Cre-mediated deletion in cardiac progenitor cells conferred by a 3'UTR-ires-Cre allele of the homeobox gene Nkx2-5.

Conditional gene targeting and transgenic strategies utilizing Cre recombinase have been successfully applied to the analysis of development in mouse embryos. To create a conditional system applicable to heart progenitor cells, a Cre recombinase gene linked at its 5' end to an internal ribosome entry site (IRES) was inserted into the 3' untranslated region of the cardiac homeobox gene Nkx2-5 using gene targeting. Nkx2-5IRESCre mice were fully viable as homozygotes. We evaluated the efficacy of Cre-mediated deletion by crossing Nkx2-5IRESCre mice with the Cre-dependent R26R and Z/AP reporter strains. Efficient deletion was observed in the cardiac crescent and heart tube in both strains. However, the Z/AP locus showed transient resistance to deletion in caudal heart progenitors. Such resistance was not evident at the R26R locus, suggesting that Cre-mediated deletion in myocardium may be locus-dependent. From cardiac crescent stages, deletion was seen not only in myocardium, but also endocardium, dorsal mesocardium and pericardial mesoderm. The Cre domain apparently includes cells dorsal to the heart that have been shown to constitute a secondary heart field, contributing myocardium to the outflow tract. Other sites of Nkx2-5 expression, including pharyngeal endoderm and its derivatives, branchial arch epithelium, stomach, spleen, pancreas and liver, also showed efficient deletion. Our data suggest that the Nkx2-5IRESCre strain will be useful for genetic dissection of the multiple tiers of lineage allocation to the forming heart as well as of molecular interactions within the heart fields and heart tube.

3' Untranslated Regions↗

PubMed1990

Direct cloning of leucine zipper proteins: Jun binds cooperatively to the CRE with CRE-BP1.

The proto-oncogene products Fos and Jun form a stable heterodimeric complex that functions in transcriptional regulation by interacting with the DNA sequence known as the AP-1 site. Dimer formation occurs through the leucine zipper, a structural motif involving a heptad repeat of leucine residues that is conserved in several fos- and jun-related genes. We have employed a novel cloning strategy to isolate genes encoding proteins capable of forming complexes with Jun. The procedure involves direct screening of a lambda gt11 cDNA library with a biotinylated Jun polypeptide. One clone isolated in this manner encodes CRE-BP1, a leucine zipper-containing protein that binds to the cyclic AMP response element (CRE) as a homodimer. CRE-BP1 also forms heterodimers with Jun but not with Fos. Jun binds cooperatively to the CRE in association with CRE-BP1. Thus, the DNA-binding specificity and affinity of Jun are modulated by association with Fos or with CRE-BP1.

Animals↗

PubMed2004

Endothelium-specific Cre recombinase activity in flk-1-Cre transgenic mice.

The use of the Cre-loxP recombination system allows the conditional inactivation of genes in mice. The availability of transgenic mice in which the Cre recombinase expression is highly cell type specific is a prerequisite to successfully use this system. We previously have characterized regulatory regions of the mouse flk-1 gene sufficient for endothelial cell-specific expression of the LacZ reporter gene in transgenic mice. These regions were fused to the Cre recombinase gene, and transgenic mouse lines were generated. In the resulting flk-1-Cre transgenic mice, specificity of Cre activity was determined by cross-breeding with the reporter mouse lines Rosa26R or CAG-CAT-LacZ. We examined double-transgenic mice at different stages of embryonic development (E9.5-E16.5) and organs of adult animals by LacZ staining. Strong endothelium-specific staining of most vascular beds was observed in embryos older than E11.5 in one or E13.5 in a second line. In addition, the neovasculature of experimental BFS-1 tumors expressed the transgene. These lines will be valuable for the conditional inactivation of floxed target genes in endothelial cells of the embryonic vascular system.

Animals↗

PubMed2002

The order of strand exchanges in Cre-LoxP recombination and its basis suggested by the crystal structure of a Cre-LoxP Holliday junction complex.

Cre recombinase uses two pairs of sequential cleavage and religation reactions to exchange homologous DNA strands between 34 base-pair (bp) LoxP recognition sequences. In the oligomeric recombination complex, a switch between "cleaving" and "non-cleaving" subunit conformations regulates the number, order, and regio-specificity of the strand exchanges. However, the particular sequence of events has been in question. From analysis of strand composition of the Holliday junction (HJ) intermediate, we determined that Cre initiates recombination of LoxP by cleaving the upper strand on the left arm. Cre preferred to react with the left arm of a LoxP suicide substrate, but at a similar rate to the right arm, indicating that the first strand to be exchanged is selected prior to cleavage. We propose that during complex assembly the cleaving subunit preferentially associates with the LoxP left arm, directing the first strand exchange to that side. In addition, this biased assembly would enforce productive orientation of LoxP sites in the recombination synapses. A novel Cre-HJ complex structure in which LoxP was oriented with the left arm bound by the cleaving Cre subunit suggested a physical basis for the strand exchange order. Lys86 and Lys201 interact with the left arm scissile adenine base differently than in structures that have a scissile guanine. These interactions are associated with positioning the 198-208 loop, a structural component of the conformational switch, in a configuration that is specific to the cleaving conformation. Our results suggest that strand exchange order and site alignment are regulated by an "induced fit" mechanism in which the cleaving conformation is selectively stabilized through protein-DNA interactions with the scissile base on the strand that is cleaved first.

Attachment Sites, Microbiological↗

PubMed2001

Self-excising retroviral vectors encoding the Cre recombinase overcome Cre-mediated cellular toxicity.

The Cre-lox system is often used to manipulate sequences in mammalian genomes. We have observed that continuous expression of the Cre recombinase in cultured cells lacking exogenous lox sites caused decreased growth, cytopathic effects, and chromosomal aberrations. Cre mutants defective in DNA cleavage were not geno- or cytotoxic. A self-excising retroviral vector that incorporates a negative feedback loop to limit the duration and intensity of Cre expression avoided measurable toxicity, retained the ability to excise a target sequence flanked by lox sites, and may provide the basis of a less toxic strategy for the use of Cre or similar recombinases.

3T3 Cells↗

PubMed1998

Maternally expressed PGK-Cre transgene as a tool for early and uniform activation of the Cre site-specific recombinase.

A transgenic mouse strain with early and uniform expression of the Cre site-specific recombinase is described. In this strain, PGK-Crem, Cre is driven by the early acting PGK-1 promoter, but, probably due to cis effects at the integration site, the recombinase is under dominant maternal control. When Cre is transmitted by PGK-Crem females mated to males that carry a reporter transgene flanked by loxP sites, even offspring that do not inherit PGK-Cre delete the target gene. It follows that in the PGK-Crem female Cre activity commences in the diploid phase of oogenesis. In PGK-Crem crosses complete recombination was observed in all organs, including testis and ovary. We prepared a mouse stock that is homozygous for PGK-Crem and at the albino (c) locus. This strain will be useful for the early and uniform induction of ectopic and dominant negative mutations, for the in vivo removal of selective elements from targeted mutations and in connection with the manipulation of targeted loci in 'knock in' and related technologies.

Animals↗

PubMed1996

Parathyroid hormone induces c-fos promoter activity in osteoblastic cells through phosphorylated cAMP response element (CRE)-binding protein binding to the major CRE.

Many parathyroid hormone (PTH)-mediated events in osteoblasts are thought to require immediate early gene expression. PTH induces the immediate early gene, c-fos, in this cell type through a cAMP-dependent pathway. The present work investigated the nuclear mechanisms involved in PTH regulation of c-fos in the osteoblastic cell line, UMR 106-01. By transiently transfecting c-fos promoter 5' deletion constructs into UMR cells, we demonstrated that PTH induction of the c-fos promoter requires the major cAMP response element (CRE). Point mutations created in the major CRE within the largest construct inhibited both PTH-stimulated and basal expression. This element, therefore, performs concerted basal and PTH-responsive cis-acting functions. Gel retardation and Western blotting techniques revealed that CRE-binding protein (CREB) constitutively binds the major CRE but becomes phosphorylated at its cAMP-dependent protein kinase consensus recognition site following PTH treatment. CREB was functionally implicated in c-fos regulation by coexpressing a dominant CREB repressor, KCREB (killer CREB), with the c-fos promoter constructs. KCREB suppressed both basal and PTH-mediated c-fos induction. We conclude that PTH activates c-fos in osteoblasts through cAMP-dependent protein kinase-phosphorylated CREB interaction with the major CRE in the promoter region of the c-fos gene.

Animals↗

PubMed2005

Tumor necrosis factor alpha (TNF) suppresses cAMP response element (CRE) activity and nuclear CRE binding protein in MA-10 mouse Leydig tumor cells.

TNF is known to suppress gonadotropin-induced steroid secretion by Leydig cells. However, the mechanisms by which this occurs are largely unknown. Because expression of many steroidogenic proteins is regulated by the PKA pathway, effects of TNF on CRE activity were examined using MA-10 mouse Leydig tumor cells. The cells were transfected with a CRE-luciferase construct, and stimulated with either LH or 8Br-cAMP in the presence or absence of TNF. TNF suppressed, LH-stimulated and 8Br-cAMP stimulated CRE activity. TNF also suppressed CRE activity stimulated with a PKA expression vector. Further experiments suggested that the effect of TNF on CRE activity was not mediated by the NF-kappaB pathway. TNF did not affect levels of either CREB or phospho-CREB in whole cell lysates; however, TNF decreased both CREB and phospho-CREB in nuclear extracts in a time-dependent manner. The decrease in nuclear CREB is likely to be a major mechanism of the suppressive effects of TNF on steroidogenesis in MA-10 Leydig cells.

Animals↗

PubMed2001

Characterization of Cre-loxP interaction in the major groove: hint for structural distortion of mutant Cre and possible strategy for HIV-1 therapy.

Although the crystal structure of Cre recombinase complexed with DNA, named loxA, was elucidated a couple of years ago, it has not yet been determined which amino acids of the protein are involved in the specific Cre-loxP interaction. Arg259 and Gln90 interact with DNA substrate in the major groove from which the specificity of protein-DNA interaction comes. In this study, we substituted these residues for other amino acids. Also, two mutated DNA substrates were constructed. In each mutant, one of the bases that interact with Arg259 or Gln90 was changed into another base. In vitro binding assays and recombination assays of variant lox sites with wild-type and mutant-type Cre revealed that Arg259 plays a key role in Cre-loxP binding but Gln90 does not. However, the recombination activity still remained intact, although the binding between Cre and DNA substrate was not ensured.

Base Sequence↗

PubMed1986

Bacteriophage P1 Cre-loxP site-specific recombination. Site-specific DNA topoisomerase activity of the Cre recombination protein.

Site-specific recombination in bacteriophage P1 occurs between two loxP sites in the presence of the Cre recombination protein. The structure of the 34-base pair loxP site consists of two 13-base pair inverted repeats separated by an 8-base pair spacer region. A mutation in the loxP site has been constructed which deletes one of the internal bases of the spacer region at the axis of dyad symmetry. This mutant loxP site shows a 10-fold reduction in recombination activity with a wild-type site both in vivo and in vitro. This low level of intramolecular recombination between a wild-type loxP site and the mutant loxP501 site is observed in vitro only when the DNA substrate is supercoiled. The majority of the supercoiled substrate is relaxed by the Cre protein, and on longer incubations, single-stranded nicks accumulate in the DNA. We have determined that these nicks occur in both the wild-type and the mutant sites. The positions of these nicks correspond to the positions of cleavage found during recombination of two wild-type sites, suggesting that the Cre protein is attempting to carry out recombination with the mutant site but most of the time this reaction is abortive. We have determined that the Cre protein relaxes a supercoiled topoisomer of a DNA substrate containing one wild-type site and one mutant site to yield a distribution of topoisomers whose linking numbers differ by steps of one, indicating that Cre can act as a type I topoisomerase.

Base Sequence↗

PubMed2000

Specificity and efficiency of Cre-mediated recombination in Emx1-Cre knock-in mice.

Emx1 is a mouse homologue of the Drosophila homeobox gene empty spiracles and its expression is restricted to the neurons in the developing and adult cerebral cortex and hippocampus. We reported previously the creation of a line of transgenic mice in which the cre gene was placed directly downstream of the putative Emx1 promoter using ES cell technology. We showed that Cre protein was present in the cerebral cortex of the transgenic mice and was able to mediate loxP-specific recombination in vitro. In the present study, the specificity and efficiency of the cre-mediated recombination were determined using three independent lines of reporter mice and a combination of histochemical staining, neuronal culture, and Southern detection of the genomic DNA. Our results showed that the recombination was highly efficient in all three lines of reporter mice tested and confirmed that the deletion was restricted to the neurons in the cerebral cortex and hippocampus. Furthermore, we have determined that the recombination efficiency in the cerebral cortex was 91%. Our results suggest that Emx1 is not expressed in every neuron in the developing and adult cerebral cortex. This line of cre mice should contribute to the studies of cortical development and plasticity.

Animals↗

PubMed1993

The trans-activator tax of human T-cell leukemia virus type 1 (HTLV-1) interacts with cAMP-responsive element (CRE) binding and CRE modulator proteins that bind to the 21-base-pair enhancer of HTLV-1.

The trans-activator protein Tax of human T-cell leukemia virus type 1 (HTLV-1) activates the viral 21-base-pair (bp) enhancer in the long terminal repeat and has been suggested to associate indirectly with the enhancer DNA. To demonstrate this, we used DNA-affinity precipitation assay and detected the Tax protein in 21-bp DNA-protein complexes isolated from HTLV-1-infected cells. To identify cellular components in the complexes, we tested various 21-bp DNA-binding proteins by gel electrophoretic mobility-shift assay. Each binding protein gave a shifted band of each 21-bp DNA-protein complex, and exogenously added Tax protein further shifted these bands of cAMP-responsive element (CRE) binding protein (CREB) and CRE modulator but did not shift other bands. Anti-Tax antibodies blocked formation of the complex, indicating complex formations of [Tax-CREB(or CRE modulator)-21-bp DNA]. The formations of these complexes paralleled the functional activities of Tax mutants. Furthermore, the Tax-CREB complex was detected in a nuclear extract of HTLV-1-infected cells, and the Tax-CREB-21-bp-DNA complex was demonstrated as a major component of Tax complexes containing the 21-bp DNA probe. These observations indicate that Tax protein binds to CREB and CRE modulator and the complexes then bind to the 21-bp enhancer, suggesting that the complex binding to the enhancer mediates trans-activation of transcription.

Cell Nucleus↗

PubMed1984

Site-specific recombination by the bacteriophage P1 lox-Cre system. Cre-mediated synapsis of two lox sites.

The bacteriophage P1-encoded recombinase Cre forms a simple DNA-protein complex at the specific recognition site loxP. Furthermore, Cre is able to mediate a synaptic union of two loxP sites. When two loxP sites are on the same linear DNA molecule, Cre binds the two sites together to form a circular protein-DNA complex. These complexes can be resolved into a linear DNA molecule and a closed circular DNA molecule, the end products of site-specific recombination.

Base Sequence↗