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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Interferon regulatory factors (IRFs) bind to the interferon-stimulated response element (ISRE) and regulate interferon- and virus-mediated gene expression. IRF-1 acts as a transcriptional activator, while IRF-2 acts as a repressor. Here we show that IRF-1 and IRF-2 bind to both cellular TFIIB, a component of the basal transcription machinery, and recombinant TFIIB (rTFIIB) and that this protein-protein interaction facilitates binding of IRFs to the ISRE. A functional interaction between TFIIB and IRF was assessed by a newly established in vitro transcription assay in which recombinant IRF-1 (rIRF-1) stimulated transcription specifically from an ISRE-containing template. With this assay we show that rIRF-1 and rTFIIB cooperatively enhance the ISRE promoter in vitro. We found that the activity of an ISRE-containing promoter was cooperatively enhanced upon cotransfection of TFIIB and IRF-1 cDNAs into P19 embryonal carcinoma cells, further demonstrating functional interactions in vivo. The cooperative enhancement by TFIIB and IRF-1 was independent of the TATA sequence in the ISRE promoter but dependent on the initiator sequence (Inr) and was abolished when P19 cells were induced to differentiate by retinoic acid treatment. In contrast, cotransfection of TFIIB and IRF-1 into NIH 3T3 cells resulted in a dose-dependent repression of promoter activation which occurred in a TATA-dependent manner. Our results indicate the presence of a cell type-specific factor that mediates the functional interaction between IRFs and TFIIB and that acts in conjunction with the requirement of TATA and Inr for promoter activation.
Mol Cell Biol 1996 Nov
PMID:Interferon regulatory factors and TFIIB cooperatively regulate interferon-responsive promoter activity in vivo and in vitro. 888 61

A polypeptide doublet (P18-P19, ca 22 kDa, pI 4.5) has been shown to accumulate in tobacco leaf plasma membrane in a development-dependent way, under constant environmental conditions. P18 and P19 were purified by 2D-PAGE and microsequenced. Microsequences revealed only small differences between the two polypeptides. A PCR-based cloning strategy identified a cDNA displaying a 591 bp ORF. The encoded polypeptide contained P19 specific microsequences. It was expressed in E. coli and a specific rabbit antiserum was raised. Western-blots confirmed its identification as P19. The accumulation pattern of hybridizable mRNA around the floral induction period was similar to that of P18 and P19. Searching of databases revealed no significant hits except unidentified plant ESTs. P18 and P19 are proposed as the first example of plant-specific and developmentally regulated plasma membrane proteins.
Biochem Mol Biol Int 1996 Oct
PMID:Cloning of a cDNA encoding a developmentally regulated 22 kDa polypeptide from tobacco leaf plasma membrane. 890 55

Retinoic acid receptor (RAR) and retinoid X receptor (RXR) form heterodimers and regulate retinoid-mediated gene expression. We studied binding of RXR- and RAR-selective ligands to the RXR-RAR heterodimer and subsequent transcription. In limited proteolysis analyses, both RXR and RAR in the heterodimer bound their respective ligands and underwent a conformational change in the presence of a retinoic acid-responsive element. In reporter analyses, the RAR ligand (but not the RXR ligand), when added singly, activated transcription, but coaddition of the two ligands led to synergistic activation of transcription. This activation required the AF-2 domain of both RXR and RAR. Genomic footprinting analysis was performed with P19 embryonal carcinoma cells, in which transcription of the RARbeta gene is induced upon retinoid addition. Paralleling the reporter activation data, only the RAR ligand induced in vivo occupancy of the RARbeta2 promoter when added singly. However, at suboptimal concentrations of RAR ligand, coaddition of the RXR ligand increased the stability of promoter occupancy. Thus, liganded RXR and RAR both participate in transcription. Finally, when these ligands were tested for teratogenic effects on zebra fish and Xenopus embryos, we found that coadministration of the RXR and RAR ligands caused more severe abnormalities in these embryos than either ligand alone, providing biological support for the synergistic action of the two ligands.
Mol Cell Biol 1997 Feb
PMID:Retinoid X receptor (RXR) within the RXR-retinoic acid receptor heterodimer binds its ligand and enhances retinoid-dependent gene expression. 900 Dec 18

Embryonal carcinoma (EC) cells can be efficiently transfected with cloned DNAs but there is a strong tendency for expression from transfected genes to be lost from stably transformed cells. To investigate the mechanism responsible for this loss of expression, we transfected P19 EC cells with a gene encoding the E. coli beta-galactosidase and examined expression of this gene in clonal populations of cells. Cells that carry and express the beta-galactosidase gene give rise to cells that do not express at a rate of about 0.02 events per cell per cell division. These non-expressing cells were of two types, some had lost the transfected genes while others had inactivated them. In those cells that retained but inactivated the transfected genes, the inactive state was stable and suppression was at the level of transcription initiation but not associated with increased DNA methylation. Because transfected DNAs integrate into the genome as tandem arrays, the gene loss and inactivation seen in EC cells may be analogous to the repeat-induced gene inactivation seen in lower eukaryotes.
Somat Cell Mol Genet 1996 Sep
PMID:Genes transfected into embryonal carcinoma stem cells are both lost and inactivated at high frequency. 903 47

ROR alpha1 and RVR are orphan members of the superfamily of nuclear hormone receptors which constitutively activate and repress, respectively, gene transcription by binding to a common DNA sequence. In an attempt to understand the physiological functions of these two transcription factors, we aimed to identify target genes. We have identified a consensus binding site for ROR alpha1 and RVR in the first intron of the N-myc gene that we designated N-myc RORE (ROR response element). Unlike most of the intronic sequence, the region encompassing the N-myc RORE is highly conserved between human and mouse, underscoring its importance. Our studies revealed that ROR alpha1 and RVR specifically bind to the human and mouse N-myc ROREs and transactivate and transrepress, respectively, reporter constructs containing the ROREs. Moreover, Northern blot analysis demonstrated a direct modulation of an exogenously introduced N-myc gene by ROR alpha1 and RVR in COS-1 cells. This effect is mediated through the N-myc RORE, since mutation of this site abolished the regulatory effects of both receptors. While transfection of ROR alpha1 in P19 embryonic carcinoma cells had no effect on the levels of endogenous N-myc mRNA, RVR down-regulated its expression. The regulatory function of the N-myc RORE was further demonstrated by the rat embryonic fibroblast (REF) transformation assay. Mutation of the RORE increased the oncogenic potential of the N-myc gene in the REF assay. The foci were more numerous and significantly larger with the mutated than with the wild-type N-myc gene, regardless of ROR alpha1 or RVR expression. Moreover, concomitant expression of ROR alpha1 and wild-type N-myc resulted in a twofold increase in the number of transformed foci. In contrast, RVR expression resulted in the formation of foci that could be established as permanent clones with a very low frequency compared to foci transformed in its absence. These observations show that ablation of the RORE results in a more oncogenic form of N-myc and suggest that deregulation of the activity of the ROR alpha1 and RVR could contribute to the initiation and progression of certain neoplasias.
Mol Cell Biol 1997 Apr
PMID:Differential regulation of the N-myc proto-oncogene by ROR alpha and RVR, two orphan members of the superfamily of nuclear hormone receptors. 912 34

The P19 embryonal carcinoma cells differentiate into neurons, astrocytes, and fibroblast-like cells following induction with retinoic acid. The cells mature into functional neurons, as determined by their ability to release neurotransmitters in a Ca(2+)- and depolarization-dependent manner. P19 neurons in culture represent a mixed population in terms of their neurotransmitter phenotype. The cholinergic phenotype of these neurons is modulated by culture density. Cholinergic markers, such as the vesicular acetylcholine transporter, acetyl cholinesterase, and choline acetyltransferase, are expressed in about 85% of the cells in sparse cultures and are largely suppressed at high cell densities. In contrast, glutamate release is enhanced in dense P19 neuronal cultures. The factor mediating the density effect is concentrated exclusively on the cell membrane of P19 neurons and not on the nonneuronal cells, which also differentiate from P19 embryonal carcinoma cells. This membrane-associated component retains its functionality, even after membrane fixation. The downregulation of the cholinergic properties in dense cultures is paralleled by a downregulation of the alpha subunit of the ciliary neurotrophic factor (CNTF) receptor. Thus, it is suggested that the membrane-associated factor, which mediates the density effect, downregulates the cholinergic phenotype by inhibiting the responsiveness of these neurons to CNTF. We further suggest that the P19 cell line can serve as a model system for the study of neurotransmitter phenotype acquisition and plasticity throughout neuronal differentiation.
J Mol Neurosci 1997 Apr
PMID:Culture density regulates both the cholinergic phenotype and the expression of the CNTF receptor in P19 neurons. 918 41

Differentiation of P19 embryonal carcinoma (EC) and embryonal stem (ES)-5 cells with retinoic acid (RA) induces expression of PTH-related peptide (PTHrP) mRNA. In this study we have characterized a region between nucleotide (nt) -88 and -58 relative to the transcription start site in the murine PTHrP gene that was involved in this expression. Sequence analysis identified two partially overlapping binding sites for the Ets family of transcription factors and an inverted Sp1-binding site. Two major specific bands were detected in a bandshift assay using an oligonucleotide spanning nt -88 and -58 as a probe and nuclear extracts from both undifferentiated and RA-differentiated P19 EC cells. The lower complex consisted of Ets-binding proteins as demonstrated by competition with consensus Ets-binding sites, while the upper complex contained Sp1-binding activity as demonstrated by competition with consensus Sp1-binding sites. The observed bandshift patterns using nuclear extracts of undifferentiated or RA-differentiated P19 cells were indistinguishable, suggesting that the differentiation-mediated expression was not caused by the induction of expression of new transcription factors. Mutations in either of the Ets-binding sites or the Sp1-binding site completely abolished RA-induced expression of PTHrP promoter reporter constructs, indicating that the RA effect was dependent on the simultaneous action of both Ets- and Sp1-like activities. Furthermore, these mutations also abolished promoter activity in cells that constitutively expressed PTHrP mRNA, suggesting a central role for the Ets and Sp1 families of transcription factors in the expression regulation of the mouse PTHrP gene.
Mol Endocrinol 1997 Sep
PMID:Expression of the parathyroid hormone-related peptide gene in retinoic acid-induced differentiation: involvement of ETS and Sp1. 928 59

Dual promoters were identified in the mouse kappa-opioid receptor (KOR) gene. The distal promoter was located in the 5'-upstream region of exon 1 and the proximal promoter was located in the first intron of this gene. The transcription initiation site of the proximal promoter was mapped to the -93rd nucleotide position from the ATG codon in a primer extension experiment. The expression of KOR mRNAs transcribed from these two promoters in mouse central nervous system and an embryonal carcinoma cell line P19 was confirmed in a ribonuclease protection assay. In non-neuronal tissues, only the transcripts initiated from the distal promoter were detected. The biological activities of these two promoters were determined in transient transfection of P19 cells with a series of reporters, each truncated at various 5'-upstream regions. It was concluded that the distal promoter was located between nucleotide positions -990 and -570, and the proximal promoter was located between nucleotide positions -330 and -93, relative to the translation initiation codon. The presence of dual promoters in the KOR gene suggested potential regulation of KOR expression by using different promoters.
Mol Pharmacol 1997 Sep
PMID:Studies of dual promoters of mouse kappa-opioid receptor gene. 928 3

Transcription of the retinoic acid receptor beta2 (RARbeta2) gene is induced by retinoic acid (RA) in mouse P19 embryonal carcinoma (EC) cells. Here we studied RA-induced chromatin structure alterations in the endogenous RARbeta2 promoter and in an integrated, multicopy RARbeta2 promoter in EC cells. RA markedly increased restriction site accessibility within the promoter, including a site near the RA responsive element (RARE) to which the nuclear receptor retinoid X receptor (RXR)-RAR heterodimer binds. These changes coincided with RA-induced alterations in the DNase I hypersensitivity pattern in and around the promoter. These changes became undetectable upon removal of RA, which coincided with the extinction of transcription. Analyses with receptor-selective ligands and an antagonist showed that increase in restriction site accessibility correlates with transcriptional activation, which parallels the RA-induced in vivo footprint of the promoter. Despite these changes, the micrococcal nuclease digestion profile of this promoter was not altered by RA. These results indicate that concurrent with the binding of the RXR-RAR heterodimer to the RARE, the local chromatin structure undergoes dynamic, reversible changes in and around the promoter without globally affecting the nucleosomal organization.
Mol Cell Biol 1997 Nov
PMID:Retinoid-induced chromatin structure alterations in the retinoic acid receptor beta2 promoter. 934 11

We have investigated the regulation of neurofilament gene expression during retinoic acid (RA)-induced neural differentiation of P19 embryonal carcinoma (EC) cells. Western blot analysis demonstrated that P19 EC cells contain significant levels of NF-L protein in the insoluble fraction but undetectable levels of NF-M and NF-H protein in either the insoluble or total cell fractions. However, immunocytochemical detection of NF-L protein in P19 EC cells showed diffuse staining within the majority of cells, rather than association with intermediate filament-like structures or staining within a subpopulation of differentiated neurons. Detectable levels of both NF-L and NF-M mRNA were present in P19 EC cells whereas NF-H mRNA remained below levels of detection, even by RT-PCR analysis. When RA-treated aggregates of P19 cells were cultured under conditions permissive for neurite outgrowth, we observed a significant increase in the amount of detectable NF-L protein localized within morphologically distinct neurons. Differentiation was also accompanied by the appearance of both the NF-M and NF-H subunits. Northern analysis revealed that this differentiation was accompanied by coincident increase in the steady-state levels of the mRNA for all three subunits and that the temporal pattern of increase was similar to what has been observed in the fetal and neonatal brain. The increase in NF-L and NF-M mRNA levels were accompanied by a concomitant increase in the rate of transcription, however, our results suggest that additional post-transcriptional mechanisms may be involved in regulating NF gene expression during the differentiation of pluripotent P19 cells.
Brain Res Mol Brain Res 1997 Oct 03
PMID:Regulation of neurofilament L, M and H gene expression during retinoic acid-induced neural differentiation of P19 embryonal carcinoma cells. 938 84


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