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Results for “Regulatory Sequences, Nucleic Acid”

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At least 19 recordsLinked to original sources

PubMed1977

Computer analysis of nucleic acid regulatory sequences.

We describe a computer program designed to facilitate the analysis of nucleic acid sequences. The program can search several nucleic acid sequences for oligonucleotides common to all of them. It can examine a DNA or RNA sequence for two kinds of homologous regions--repetitions and dyad symmetries. The homologies need not be perfect: mismatches and "looping out" of nucleotides are allowed. The program also finds (A+T)- and (G+C)-rich regions, locates restriction enzyme recognition sites, determines the distribution of di- and trinucleotides, and performs various other functions. We include two representative applications of the program. All published prokaryotic transcription termination sequences (June 1977) were found to share the following features: (i) a string of at least five T residues, (ii) the sequence CGGGC or a close analog immediately preceding the T cluster, (iii) a region of strong dyad symmetry preceding the Ts and including the CGGGC sequence. A sequence of 221 nucleotides consisting of the Escherichia coli trp promoter, operator, and leader was found to contain two strong dyad symmetries. These homologies both occur at known regulatory sites; no comparable homologies occur in regions without regulatory significance.

Base Sequence↗

PubMed2004

DNA-looping by RXR tetramers permits transcriptional regulation "at a distance".

RXR, a member of the superfamily of nuclear hormone receptors, regulates gene transcription in response to 9-cis-retinoic acid. We previously showed that, among nuclear receptors, RXR is unique in that it self-associates into homotetramers, and that these tetramers dissociate rapidly upon ligation. Here, we report that binding of RXR tetramers to DNA containing two RXR response elements results in a dramatic DNA-looping. RXR can thus juxtapose distant DNA sequences, enabling transcriptional regulation by far-upstream factors. We show that RXR functions as a DNA architectural factor and that, while this activity is regulated by 9-cis-retinoic acid, it is distinct from and independent of the receptor's intrinsic transcriptional activity. The data establish RXR as the first identified architectural factor whose activity is regulated by a small ligand, and demonstrate a novel mechanism of transcriptional regulation by retinoids.

Animals↗

PubMed1994

Regulation of intercellular adhesion molecule-1 expression by retinoic acid: analysis of the 5' regulatory region of the gene.

Human intercellular adhesion molecule-1 (ICAM-1), a specific ligand for the lymphocyte-function-associated antigen-1, plays an important role in immune responses. ICAM-1 expression is regulated by various proinflammatory cytokines, by PMA, and by retinoic acid. In this study, we have investigated the mechanisms of transcriptional control involved in the stimulation of ICAM-1 gene expression by retinoic acid in SK-N-SH cells. Northern-blot analysis demonstrated that ICAM-1 mRNA is maximally induced at 24 hr, suggesting that it is not an early-response gene with respect to retinoic-acid responsiveness, whereas the retinoic acid receptor-beta mRNA level was maximal 12 hr following retinoic acid treatment. To analyze the 5'-regulatory region of the ICAM-1 gene, an EcoRI/SaII fragment spanning the first 1.3 kb upstream of the translational start site was used to direct the expression of a linked luciferase reporter gene in transient transfection assays in SK-N-SH cells. A 24-hr treatment of transfected cells with 10 microM retinoic acid resulted in a 10- to 13-fold increase in luciferase activity compared with untreated cells. Deletion mutant analysis revealed that a region located between -393 and -176 bp from the translational start site is critical for retinoic acid stimulation of luciferase activity. This region harbors a consensus sequence for a retinoic-acid-responsive element (RARE) homologous to the element found upstream of the alcohol dehydrogenase-3 gene. Co-transfection of expression vectors encoding the retinoic acid receptor-alpha, -beta, or -gamma, with reporter plasmids harboring the putative RARE, confirmed that the ICAM-1 gene is regulated by retinoic acid in a retinoic acid receptor-dependent fashion.

Base Sequence↗

PubMed1990

Negative regulation of the rat stromelysin gene promoter by retinoic acid is mediated by an AP1 binding site.

Stromelysin is a member of the metalloproteinase family which plays an important role in extracellular matrix remodelling during many normal and disease processes. We show here that in polyomavirus-transformed rat embryo fibroblast cells (PyT21), the transcription from the stromelysin gene is repressed by the vitamin A derivative retinoic acid (RA). Furthermore, expression vectors encoding the human RA receptors hRAR-alpha, hRAR-beta and hRAR-gamma repress chloramphenicol acetyltransferase (CAT) expression from stromelysin promoter-CAT gene expression vectors in RA-treated PyT21 and human HeLa cells, as determined by transient transfection assays. Through mutation and deletion analysis, we show that the RA dependent repression is mediated by a 25 bp region from nucleotide positions -72 to -48 of the rat stromelysin 5'-flanking DNA sequence. Further mutation analysis of this region indicates that the DNA sequence required for RA dependent repression colocalizes with an AP1 binding site which is essential for promoter activity. We show also that RA represses the transcriptional activity of a reporter gene containing a TPA responding AP1 binding site driving the HSV tk promoter. Thus the RAR-RA complex appears to repress transcription of the stromelysin gene by blocking activation by positive regulatory factors. However, we found no evidence supporting the possibility that the RA dependent repression could be due to RAR binding to the AP1 binding site or to the AP1 components c-fos and c-jun.

Animals↗

PubMed1991

A retinoic acid response element is present in the mouse cellular retinol binding protein I (mCRBPI) promoter.

Genomic and cDNA sequences for the mouse cellular retinol binding protein I (mCRBPI) are presented. A specific cis-acting element responsible for retinoic acid (RA) inducibility of the mCRBPI promoter was identified and characterized. Deletion mapping of a CRBPI promoter--chloramphenicol acetyltransferase reporter gene construct localized this element to a 259 bp restriction fragment located approximately 1 kb upstream from the transcription start-site. A sequence closely resembling the previously characterized RA response element (RARE) of the RA receptor beta 2 (RAR-beta 2) promoter, and consisting of a direct repeat of the motif 5'-GGTCA-3' separated by three nucleotides, was found within this restriction fragment. Mutation of these 5'-GGTCA-3' motifs to GGAGC and GGGGC abolished RA-inducible transcription whereas a mutation to a direct repeat of the GTTCA motif found in the RARE of the RAR-beta 2 promoter resulted in enhanced inducibility. Oligonucleotides containing the direct repeat of the GGTCA motif were able to confer RA-dependent transcriptional enhancement to the herpes simplex thymidine kinase promoter, as well as to bind directly all three retinoic acid receptors (RARs) alpha, beta and gamma, as determined by gel retardation/shift assays. The control of CRBPI gene transcription by RA-RAR complexes interacting with the RARE characterized here may correspond to a feedback mechanism important in regulating retinoid metabolism and action.

Amino Acid Sequence↗

PubMed1991

The late retinoic acid induction of laminin B1 gene transcription involves RAR binding to the responsive element.

Transcription of the murine laminin B1 (LB1) gene is induced by retinoic acid (RA), but responds only 24-28 h after RA treatment in F9 EC cells. Here we have shown by gel retardation assay that all three retinoic acid receptors (RARs) alpha, beta and gamma expressed in Cos cells can bind directly to the previously characterized retinoic acid response element (RARE) of the LB1 promoter, albeit with a weaker affinity than to the RAR-beta gene RARE. Three stereo-aligned TGACC-like motifs are crucial for this binding. Interestingly, the capacity of RAR-alpha, -beta and -gamma to bind the LB1 RARE appears to be differentially modulated by factor(s) present in HeLa cells infected with RAR-expressing vaccinia virus vectors. Analyses of LB1 RARE mutants provide a strong correlation between RA-inducibility in vivo and efficiency of RAR binding in vitro. Thus, RARs can participate directly in transcriptional induction of the LB1 gene, even though this induction is cycloheximide sensitive and RARs are present in F9 cells prior to RA addition.

Base Sequence↗

PubMed2001

Regulation of male sexual development by Sry and Sox9.

Sry, a gene from the Y chromosome, is known to initiate testis formation and subsequent male differentiation in mammals. A related gene, Sox9, also plays a critical role in testis determination, possibly in all vertebrates. A number of models have been presented regarding the molecular modes of action of these two genes. However, details regarding their regulation, regulatory target genes, and interacting protein factors and co-factors have not been established with any certainty. In this review, we examine new evidence and re-examine existing evidence bearing on these issues, in an effort to build up an integrative model of the network of gene activity centred around Sry and Sox9. J. Exp. Zool. 290:463-474, 2001.

Animals↗

PubMed2001

Modulation of the expression of peroxisome proliferator-activated receptor-dependent genes through disproportional expression of two subtypes in the small intestine.

We have reported that dietary long-chain triacylglycerols (LCT) enhance the transcription of cellular retinol-binding protein, the type II (CRBPII) gene, and the liver-type fatty acid-binding protein (L-FABP) gene in the small intestine. Because the cis elements on the CRBPII gene consisting of two AGGTCA motifs separated by a single nucleotide are known to bind not only the 9-cis-retinoic acid receptor (RXR) homodimer, but also the peroxisome proliferator-activated receptor (PPAR)-RXR heterodimer, it has been implicated that the unsaturated long-chain fatty acids, as the ligands of the PPAR, might activate the transcription of the CRBPII gene, thereby making use of the RXR-response elements (RXRE and RE3) as the PPAR-response element (PPRE). In this study, we found that the PPARalpha mRNA level in the rat jejunum was elevated by dietary fat, whereas the PPARdelta mRNA level was reduced under this condition. Electrophoretic mobility-shift assay revealed that both PPARalpha-RXRalpha and PPARdelta-RXRalpha heterodimers, specifically and in a dose-dependent manner, bound to the two PPRE-like elements of the rat CRBPII gene as well as the known PPREs in the L-FABP and acyl-CoA oxidase genes. The binding of the PPARalpha-RXRalpha heterodimer to the CRBPII-RXRE, the CRBPII-RE3, and the PPREs of L-FABP, HMG-CoA synthase, and acyl-CoA oxidase was gradually diminished by the addition of increasing amounts of PPARdelta. The binding of the PPARdelta-RXRalpha heterodimer to CRBPII-RXRE, CRBPII-RE3, and other PPREs was also gradually reduced by the addition of increasing amounts of PPARalpha. Using Escherichia coli-expressed RXRalpha, we showed that the mutual competition for RXRalpha with PPARalpha and PPARdelta occurred at the protein level. These results suggest that the transcriptions of CRBPII, L-FABP, and the other PPAR-dependent genes in the small intestine may be coordinately regulated by the disproportional expression of PPARalpha and PPARdelta.

Animals↗

PubMed1993

PPAR-RXR heterodimer activates a peroxisome proliferator response element upstream of the bifunctional enzyme gene.

A DNA sequence that confers a response to a class of rodent hepatocarcinogens termed peroxisome proliferators has been identified 2947bp upstream of the rat peroxisomal bifunctional enzyme gene. Two members of the steroid hormone receptor family, termed the peroxisome proliferator activated receptor (PPAR alpha) and the retinoid X receptor (RXR alpha), co-operate to bind specifically to this sequence. Importantly, this response element (PPRE) is similar to that identified upstream of other peroxisome proliferator responsive genes such as those encoding acyl CoA oxidase and cytochrome P450 IVA6. These data therefore provide further evidence that PPAR alpha plays an important role in mediating the action of peroxisome proliferators.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1994

cis-acting elements in 5'-flanking region of rat alpha-fetoprotein mediating retinoic acid responsiveness.

A distal RA responsive cis-acting element has been identified in the 5'-flanking region of the alpha-fetoprotein gene by transfection of different deletion mutants of AFP-CAT fusion gene. The retinoic acid receptor specifically binds to this RA responsive cis-acting element in mobility shift assays. Furthermore, this cis-acting element functions in exogenous TK promoter in transient cotransfection assays. This study suggests a role for the RA responsive cis-acting element in the RA induction of alpha-fetoprotein gene expression.

Animals↗

PubMed1995

Effects of vitamin A deficiency and retinoic acid treatment on expression of a phosphoenolpyruvate carboxykinase-bovine growth hormone gene in transgenic mice.

Vitamin A regulation of specific promoter domains of the phosphoenolpyruvate carboxykinase (PEPCK) gene was tested in a PEPCK/bovine growth hormone (bGH) transgenic mouse model. Vitamin A deficiency causes a significant decrease in hepatic bGH mRNA when expression is driven by either a 533-base-pair (bp) PEPCK promoter fragment (from position -460 to +73) or a 428-bp PEPCK promoter fragment (from position -355 to +73). Treatment of vitamin A deficient transgenic mice with all-trans retinoic acid (RA) increases bGH mRNA levels above those measured with the deficiency. Hepatic retinoic acid receptor (RAR)beta mRNA levels also change with vitamin A deficiency and supplementation, but not RAR alpha mRNA levels. These results indicate that all-trans RA plays a physiologic role in regulating expression of a gluconeogenic gene in liver.

Animals↗

PubMed1999

Differential regulation of ligand-dependent and ligand-independent functions of the mouse retinoid X receptor beta by alternative splicing.

RXR is involved in two distinct signal transduction pathways. RXR activates transcription ligand-dependently on RXRE and behaves as a positive heterodimer partner with permissive receptors; while it behaves as a ligand-independent silent partner with nonpermissive receptors. We studied the functions of a unique isoform of mouse RXRbeta2 (mRXRbeta2E) which has an in-frame four-amino-acid insertion in the ligand binding domain. mRXRbeta2E did not bind 9-cis RA with high affinity and did not activate transcription on RXRE. However, mRXRbeta2E formed heterodimers with TR, VDR, RAR, and LXR, and enhanced transcriptional activity of the nonpermissive VDR to the same extent as mRXRbeta2. With the permissive LXR, mRXRbeta2E did not show 9-cis RA-dependent transactivation and inhibited mRXRbeta2 activity. Thus, although mRXRbeta2E can perform ligand-independent functions of RXR, it cannot mediate the ligand-dependent functions of RXR, indicating that alternative splicing plays an important role in the differential regulation of these two important functions of RXR.

Alitretinoin↗

PubMed1995

Transcriptional modulation of the human intercellular adhesion molecule gene I (ICAM-1) by retinoic acid in melanoma cells.

Retinoids play an important role as differentiating agents in a variety of normal and neoplastic cells and have been reported to induce ICAM-1 levels in melanomas, a phenomenon that we confirm in this paper. The effects of retinoids on gene expression usually involve the binding of specific retinoic acid receptor trans-acting factors (RARs) with their ligands, which then interact with specific target sites, the retinoic acid responsive elements (RAREs) present in the promoters of responsive genes. In the case of ICAM-1, we have cloned and analyzed the proximal regulatory region of the human gene. We show that the ICAM-1 promoter is RA-inducible, that it contains a putative consensus RARE (GGGTCATCGCCCTGCC), which binds in vitro RAR alpha complemented with RXRs, and that mutation of the RARE abrogates promoter responsiveness to RA. These studies allow ICAM-1 to be added to the list of genes transcriptionally activated by RA acting through an RARE element.

Base Sequence↗

PubMed1994

The promoter of the H1zero histone gene contains a DNA element bound by retinoic acid receptors.

Retinoic-acid mediated differentiation of F9 cells is accompanied by an increased transcription of the histone H1zero gene. This increase is an early response after addition of retinoic acid, suggesting a direct effect of the hormone on transcription of the gene. We show now that the promoter of histone H1zero contains a DNA element, localized 531 base-pairs upstream of the cap site, that is composed of a direct repeat of the sequence PuGGTCA separated by eight base-pairs. This element confers retinoic acid responsiveness to a heterologous thymidine kinase promoter in F9 and HeLa cells. Furthermore, the element forms retarded complexes not only with bacterially expressed retinoic acid receptors (RARs) and retinoid X receptors (RXRs), but also with endogenous F9 receptors. Our results suggest therefore that retinoic acid receptors can control the expression of a chromatin structural gene the expression of which is associated with a differentiated phenotype.

Animals↗

PubMed1997

Genomic footprinting of retinoic acid regulated promoters in embryonal carcinoma cells.

Retinoic acid (RA) treatment of embryonal carcinoma (EC) cells initiates a cascade of alterations in gene regulation, leading to their differentiation into various cell types. In P19 EC cells RA treatment stimulates induction of the RAR beta gene, while it represses Oct3/4 gene expression. Here we present dimethylsulfate-based genomic footprinting analyses of these two genes. We found that the RAR beta promoter is not occupied prior to RA treatment, but following RA treatment all regulatory elements in this promoter become occupied. On the other hand, the Oct3/4 promoter is occupied at all three known elements before RA treatment, but this occupancy is coordinately lost following the treatment. Thus, factor occupancy coincides with expression of the genes. It is likely that the presence of factor binding or its absence revealed here represents a mechanism of the regulated expression of these genes in vivo. Our results demonstrate the power of genomic footprinting for studying regulatory events for transcription in vivo. In contrast, with in vitro protein-DNA binding assay, factors for both promoters are present in these cells regardless of RA treatment. It has been shown that RA receptor (RAR) and retinoid X receptor (RXR), by heterodimerization, mediate the RA action in EC cells. To elucidate the role of RAR/RXR heterodimers in the RAR beta promoter occupancy in vivo, genomic footprinting has been performed in P19 cells stably expressing dominant negative mutants of RXR. Two such mutants, lacking either the DNA binding domain or the C-terminal activation domain, inhibit RA induction of the RAR beta gene in these cells. RA-induced factor occupancy is also markedly inhibited at all elements in the RAR beta promoter in these cells. Our results show that binding of liganded RAR/RXR heterodimers to RARE is required for other factors to gain access to their respective elements in the promoter.

Animals↗

PubMed1999

Retinoid receptors in health and disease: co-regulators and the chromatin connection.

Retinoid receptors behave as ligand-dependent transcriptional regulators, repressing transcription in the absence of ligand and activating transcription in its presence. The different effects on transcription are carried out through recruitment of co-regulators: unliganded receptors bind corepressors (NCoR and SMRT) that are found within a complex containing histone deacetylase (HDAC) activity, whereas liganded receptors recruit coactivators with histone acetylase activity (HATs). Chromatin remodeling activities have also shown to be required, suggesting a hierarchy of promoter structure modifications in RA target genes carried out by multiple coregulatory complexes. In this review, we examine the experimental evidence for the model just sketched. We focus on recent findings highlighting new molecular details in receptor-coregulator interactions, including the discovery and initial characterisation of novel complexes with multiple chromatin modifying activities. Finally, we look at the role of aberrant recruitment of the NCoRHDAC complex by altered retinoid receptors in the pathogenesis of acute promyelocytic leukemia. These results point to a crucial role for control of transcription factor coregulator interactions in the regulation of cellular processes, and suggest new molecular targets for cancer therapy.

Acetylation↗

PubMed1999

Sry and Sox9: mammalian testis-determining genes.

Sry is the Y-chromosomal gene that acts as a trigger for male development in mammalian embryos. This gene encodes a high mobility group (HMG) box transcription factor that is known to bind to specific target sequences in DNA and to cause a bend in the chromatin. DNA bending appears to be part of the mechanism by which Sry influences transcription of genes downstream in a cascade of gene regulation leading to maleness, but the direct targets of Sry remain to be positively identified. One gene known to be downstream from Sry in this cascade is Sox9, which encodes a transcription factor related to Sry by the HMG box. Like Sry, mutations in Sox9 disrupt male development, but unlike Sry, the role of Sox9 is not limited to mammals. This review focuses on what is known about the two genes and their likely modes of action, and draws together recent data relating to how they might interconnect with the network of gene activity implicated in testis determination in mammals.

Amino Acid Sequence↗

PubMed1992

Retinoic acid-induced changes in differentiation-defective embryonal carcinoma RAC65 cells.

RAC65 is a mutant clone of mouse embryonal carcinoma cells, P19, which does not undergo terminal differentiation upon treatment with retinoic acid (RA). RAC65 cells express a truncated RA receptor alpha (RAR alpha) which, however, does not fully explain their defect. Here we show that RAC65 cells exhibit an additional defect in RAR alpha mRNA which may reflect a defect in RNA splicing. The parental and mutant cells also differ in their capacities to bind [3H]RA into nuclear fractions and in expression of cellular RA binding protein (CRABP) mRNA after treatment with RA. The combined data suggest that the defect in RAC65 RAR alpha results in reduced expression of the CRABP gene after RA treatment and, therefore, increased flow of RA into the nucleus.

Animals↗