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Results for “regulatory element transcripts”

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PubMed2000

Identification of two regulatory elements within the high mobility group box transcription factor XTCF-4.

Some members of the Wnt family of extracellular glycoproteins regulate target gene expression by inducing stabilization and nuclear accumulation of beta-catenin, which functions as a transcriptional activator after binding to transcription factors of the T-cell factor/lymphoid enhancer factor (TCF/LEF) family. Three different members of this family have been identified in Xenopus laevis thus far that differ in their ability to influence mesodermal differentiation and to activate expression of the Wnt target gene fibronectin. Here we report on the isolation and characterization of additional variants of XTCF-4. We show that the differential ability of these proteins and other members of the TCF family to activate target genes is neither due to preferential interaction with transcriptional cofactors of the groucho family or SMAD4 nor to different DNA binding affinities. Expression of these proteins in an epithelial cell line reveals differences in their ability to form a ternary complex with DNA and beta-catenin. Interestingly, formation of this ternary complex was not sufficient to activate target gene expression as previously thought. Our experiments identify two amino acid sequence motifs, LVPQ and SFLSS, in the central domain of XTCF-4 that regulate the formation of the DNA-TCF-beta-catenin complex or activation of target genes, respectively. Biochemical studies reveal that the phosphorylation state of these XTCF-4 variants correlates with their ability to form a ternary complex with beta-catenin and DNA but not to activate target gene expression. The described variants of XTFC-4 with their different properties in complex formation provide strong evidence that in addition to the regulation of beta-catenin stability the isoforms of TCF/LEF transcription factors and their posttranslational modifications define the cellular response of a Wnt/wingless signal.

Amino Acid Sequence↗

PubMed2026

Genetic Deletion of Cis-Regulatory Elements to Dissect the Function of the Non-coding Genome in human Preimplantation Models.

Cis-regulatory elements coordinate gene expression in a spatially and temporally controlled manner and contribute to the establishment of distinct cellular states during development. A substantial proportion of transcriptionally active cis-regulatory elements in primate embryos originated from ancient retroviral integrations into the germline. These endogenous retroviruses, also known as long terminal repeat retrotransposons, retain intrinsic regulatory activity and are often species-specific, making them strong candidates for regulating species-divergent aspects of embryonic development. Ethical and legal restrictions on human embryo research have historically limited direct investigation of gene regulation during human embryogenesis. Human naive pluripotent stem cells and three-dimensional stem cell-based blastocyst models provide alternative systems for studying early developmental processes. This protocol describes the CRISPR-Cas9-mediated deletion of endogenous retrovirus-derived cis-regulatory elements in human naive pluripotent stem cells. Preassembled Cas9 and single-guide RNA ribonucleoprotein complexes are delivered by nucleofection, followed by single-cell cloning, PCR-based genotyping, Sanger sequencing, expansion, cryopreservation, and genomic stability assessment of the edited lines. The resulting wild-type, heterozygous, and homozygous or hemizygous deletion clones provide a platform for investigating the contribution of individual endogenous retrovirus-derived elements to gene regulation in human preimplantation models. This method enables direct functional interrogation of species-specific non-coding regulatory sequences and supports the study of transcriptional mechanisms involved in early human development.

Humans↗

PubMed2003

The variant hepatocyte nuclear factor 1 activates the P1 promoter of the human alpha-folate receptor gene in ovarian carcinoma.

The alpha folate receptor (alpha FR) is a membrane glycoprotein that binds folates, and mediates their uptake and that of antifolate drugs. alpha FR is absent on ovarian surface epithelium (OSE) but is detectable during early transforming events in this epithelium, with increasing expression levels in association with tumor progression. Analysis of transcriptional regulation of the alpha FR gene have revealed two promoter regions, P1 and P4, flanking exons 1 and 4, respectively, and a requirement for three SP1 sites and an INR element for optimal P4 activity. Here, we focused on the P1 transcription regulation in ovarian carcinoma cells. RNase protection assay indicated that the 5'-untranslated region is heterogeneous because of different start sites and alternative splicing of exon 3. A core region of the P1 promoter was sufficient for maximal promoter activity in ovarian carcinoma cell lines but not in OSE cells or in alpha FR-nonexpressing cell lines. Deletion and mutation analysis of this core promoter identified a cis-regulatory element at position +27 to +33 of the untranslated exon 1, which is responsible for maximum P1 activity. This element formed an abundant DNA-protein complex with nuclear proteins from ovarian cancer cells but not from other cell lines or OSE cells. Competition experiments and supershift assays demonstrated binding of the P1 cis-regulatory element by a transcription factor involved in embryonic development, the variant hepatocyte nuclear factor-1 (vHNF1). Analysis of RNA from various cell lines and surgical specimens confirmed that vHNF1 is expressed in ovarian carcinomas. Thus, vHNF1 regulates tissue-specific transcription in ovarian carcinoma.

5' Untranslated Regions↗

PubMed1994

beta-Casein mRNA sequesters a single-stranded nucleic acid-binding protein which negatively regulates the beta-casein gene promoter.

beta-Casein gene expression in mammary epithelial cells is under the control of the lactogenic hormones, glucocorticoids, insulin, and prolactin. The hormonal control affects gene transcription, and several regulatory elements in the beta-casein gene promoter between positions -80 and -221 have previously been identified. A region located in the promoter between positions -170 and -221 contains overlapping sequences for negative and positive regulatory elements. A sequence-specific single-stranded DNA-binding factor (STR), composed of two proteins with molecular masses of 35 and 54 kDa, recognizes the upper strand of this region and has a repressing role in transcription. High-level STR binding activity was observed in nuclear extracts from mammary glands of pregnant and postlactating mice and from noninduced HC11 mammary epithelial cells, cells with a low level of transcriptional activity of the beta-casein gene. STR activity is downregulated in mammary epithelial cells during lactation of the animals and after lactogenic hormone induction of HC11 cells in culture. These cells strongly transcribe the beta-casein gene. We investigated the mechanism of downregulation and found that a lactogenic-hormone-induced molecule (I-STR) inhibits STR from binding to its DNA target. I-STR is composed of RNA. STR is sequestered into the cytoplasm by I-STR after lactogenic hormone induction of mammary epithelial cells and remains present in an RNA-bound form. A high-affinity STR binding site was found in the 5' untranslated region of beta-casein mRNA. We propose that beta-casein mRNA can function as I-STR. beta-Casein mRNA may positively regulate its own transcription by translocating STR from the nucleus to the cytoplasm. The beta-casein STR binding sequence increases expression of a transfected beta-galactosidase gene when it is placed into the 5' untranslated region sequence of the mRNA. STR may have a positive role in posttranscriptional regulation.

Animals↗

PubMed2002

Charge movement and transcription regulation of L-type calcium channel alpha(1S) in skeletal muscle cells.

Several factors, such as Ca(2+), trophic factors and ageing, regulate dihydropyridine-sensitive receptor (DHPR) alpha(1) subunit expression. However, basic mechanisms of DHPR alpha(1S) expression are unknown. To better understand the regulatory elements that control transcription, the 1.2 kb 5'-flanking region fragment immediately upstream of the mouse L-type Ca(2+) channel or DHPR alpha(1S) gene was isolated and sequenced. Luciferase reporter constructs driven by different promoter regions of mouse DHPR alpha(1S) gene were used for transient transfection assays in muscle C2C12 cells. In these preparations we found that three regions corresponding to CREB, GATA-2 and SOX-5 consensus sequence within the 5'-flanking region of the DHPR alpha(1S) gene are important for DHPR alpha(1S) gene transcription. Antisense oligonucleotides against CREB, GATA-2 and SOX-5 significantly reduced charge movement in C2C12 cells. Charge movement was recorded in the whole-cell configuration of the patch clamp technique. Results from cells transfected with antisense (AS) and sense (S) oligonucleotides and nontransfected cells were compared. Charge movement experiments were fitted to a Boltzmann equation. Maximum charge movement (Q(max)) (nC microF(-1), mean +/- S.E.M.) for S- and AS-CREB was 70.3 +/- 2.9 and 52.8 +/- 3.3, respectively (P < 0.05). The same parameter for S- and AS-GATA-2 was 71.3 +/- 3.9 and 48.2 +/- 2.3, respectively (P < 0.05) and for S- and AS-SOX-5 was 70.4 +/- 4.2 and 45.1 +/- 3.2, respectively (P < 0.05). Values recorded in cells transfected with sense S-CREB, S-GATA-2 and S-SOX-5 oligonucleotides were not significantly different from those recorded in nontransfected cells. This study demonstrates that the transcription factors CREB, GATA-2 and SOX-5 play a significant role in the expression of the skeletal muscle DHPR or L-type Ca(2+) channel alpha(1S).

Animals↗

PubMed1990

Decoding positional information: regulation of the pair-rule gene hairy.

In the series of local gene activations that occur during early Drosophila development, the striped expression patterns of the pair-rule genes provide the first indication of segmental periodicity. The experiments that we report here address the question of how these patterns arise, by studying the regulation of one of these genes, hairy. We show that each of the seven stripes of hairy expression is controlled by a distinct subset of cis-acting regulatory elements, some mediating transcriptional activation and others transcriptional repression. In general, elements necessary and sufficient for triggering a particular stripe response are clustered on the DNA and appear to overlap or be interspersed with elements involved in at least one other stripe response. Our results extend previous findings suggesting that periodic hairy expression arises by a decoding process in which each stripe is triggered by particular combinations or concentrations of regulatory factors. These regulatory factors are likely to include the products of the gap class of segmentation genes that are required for activating or positioning particular subsets of hairy stripes and are expressed with overlapping distributions during early embryogenesis.

Animals↗

PubMed1989

An internal regulatory element controls troponin I gene expression.

During skeletal myogenesis, approximately 20 contractile proteins and related gene products temporally accumulate as the cells fuse to form multinucleated muscle fibers. In most instances, the contractile protein genes are regulated transcriptionally, which suggests that a common molecular mechanism may coordinate the expression of this diverse and evolutionarily unrelated gene set. Recent studies have examined the muscle-specific cis-acting elements associated with numerous contractile protein genes. All of the identified regulatory elements are positioned in the 5'-flanking regions, usually within 1,500 base pairs of the transcription start site. Surprisingly, a DNA consensus sequence that is common to each contractile protein gene has not been identified. In contrast to the results of these earlier studies, we have found that the 5'-flanking region of the quail troponin I (TnI) gene is not sufficient to permit the normal myofiber transcriptional activation of the gene. Instead, the TnI gene utilizes a unique internal regulatory element that is responsible for the correct myofiber-specific expression pattern associated with the TnI gene. This is the first example in which a contractile protein gene has been shown to rely primarily on an internal regulatory element to elicit transcriptional activation during myogenesis. The diversity of regulatory elements associated with the contractile protein genes suggests that the temporal expression of the genes may involve individual cis-trans regulatory components specific for each gene.

Animals↗

PubMed1997

Modulation of erythropoietin delivery from engineered muscles in mice.

In most relevant diseases, the permanent systemic delivery of a therapeutic protein from engineered cells might be proposed only if secretion levels can be regulated. The tetracycline resistance operon of Escherichia coli provides a transcriptional regulatory system effective in mammalian cells, which could be used for that purpose. A chimeric transactivator (tTA) consisting of the tetracycline repressor fused to the transactivation domain of the herpes simplex virus VP16 protein stimulates transcription by binding a minimal cytomegalovirus (CMV) promoter containing repeats of the tetracycline operator (tetO-CMV). Binding is abolished by tetracycline, thus impairing promoter activation. We have transduced C2.7 myoblasts with two U3-deleted retroviral vectors containing these regulatory elements. The tetP-Epo vector expressed the murine erythropoietin (Epo) cDNA under the control of the tetO-CMV promoter. The D-De-tTA vector expressed tTA under the control of the muscle-specific human desmin enhancer-promoter. Northern blot analysis showed background Epo mRNA expression in myoblasts. Myotubes differentiation induced tTA expression, leading to a 28-fold increase of Epo mRNAs, which was suppressed by tetracycline. Basal Epo secretion in myoblasts increased 23- to 41-fold during the formation of multinucleated myotubes, and turned back close to myoblast level when tetracycline was added. Myoblasts transduced with both vectors and treated with mitomycin with the aim to prevent tumor formation were engrafted in skeletal muscles of syngeneic C3H mice. Hematocrit levels were significantly higher in animals bearing cells transduced with both vectors than in control animals grafted with cells transduced with the Epo vector only. This difference was abolished when tetracycline was given to mice. These data indicate that the tetracycline regulatory elements can modulate transcription in the context of retroviral vector genomes, and that this system can be used to control the in vivo delivery of a therapeutic protein from genetically modified muscles.

Animals↗

PubMed2002

A GC-box within the proximal promoter region of the rat cathepsin L gene activates transcription in Sertoli cells of sexually mature rats.

It has been proposed that stage-specific gene expression in Sertoli cells results from sequential activation and repression of transcription. However, the exact molecular mechanisms are unknown. As a first step in addressing this fundamental issue, we recently demonstrated that a 3-kilobase (kb) genomic fragment immediately upstream of the rat cathepsin L translation start site directed stage-specific expression of a reporter gene only in Sertoli cells of transgenic mice in a manner comparable to that of the endogenous gene (predominantly in stages VI-VIII tubules). Supporting the activation/repression model of regulation, an upstream domain that mediated an inhibitory effect by male germ cells was identified within this 3-kb promoter region. In the present study, we localized and characterized the regulatory elements that activate transcription. Analyses of a series of 5' deletion constructs demonstrated that a 120-base pair (bp) region that spans the transcription start site of the rat cathepsin L gene was sufficient to activate transcription in Sertoli cells isolated from sexually mature rats. Within this region, electrophoretic mobility shift assays showed that one member of the Sp/XKLF family of factors, Sp3, specifically bound to a GC-box. Furthermore, Sp1-binding activity was not detected in nuclear extracts from Sertoli cells of sexually mature rats. Finally, the GC-box was shown to be essential for promoter activity since mutating this binding motif abolished promoter activity. Collectively, these results suggest that the GC-box is a critical regulatory element for the cathepsin L promoter in mature Sertoli cells.

Animals↗

PubMed1992

Two regulatory proteins that bind to the basic transcription element (BTE), a GC box sequence in the promoter region of the rat P-4501A1 gene.

The cDNAs for two DNA binding proteins of BTE, a GC box sequence in the promoter region of the P-450IA1(CYP1A1) gene, have been isolated from a rat liver cDNA library by using the BTE sequence as a binding probe. While one is for the rat equivalent to human Sp1, the other encodes a primary structure of 244 amino acids, a novel DNA binding protein designated BTEB. Both proteins contain a zinc finger domain of Cys-Cys/His-His motif that is repeated three times with sequence similarity of 72% to each other, otherwise they share little or no similarity. The function of BTEB was analysed by transfection of plasmids expressing BTEB and/or Sp1 with appropriate reporter plasmids into a monkey cell line CV-1 and compared with Sp1. BTEB and Sp1 activated the expression of genes with repeated GC box sequences in promoters such as the simian virus 40 early promoter and the human immunodeficiency virus-1 long terminal repeat promoter. In contrast, BTEB repressed the activity of a promoter containing BTE, a single GC box of the CYP1A1 gene that is stimulated by Sp1. When the BTE sequence was repeated five times, however, BTEB turned out to be an activator of the promoter. RNA blot analysis showed that mRNAs for BTEB and Sp1 were expressed in all tissues tested, but their concentrations varied independently in tissues. The former mRNA was rich in the brain, kidney, lung and testis, while the latter was relatively abundant in the thymus and spleen.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2003

Transcriptional regulation of the CRLR gene in human microvascular endothelial cells by hypoxia.

Adrenomedullin is a 52 amino acid peptide that shows a remarkable range of effects on the vasculature that include inter alia, vasodilatation, regulation of permeability, inhibition of endothelial cell apoptosis, and promotion of angiogenesis. Recently the G-protein coupled receptor (GPCR) calcitonin receptor-like receptor (CRLR), and receptor activity modifying proteins (RAMPs) have become recognized as integral components of the adrenomedullin signaling system. However, mechanisms of regulation of CRLR expression are still largely unknown. This is in part due to lack of information on the gene promoter. In this study we have determined the transcriptional start of human CRLR cDNA by 5'-RACE and cloned the proximal 5'-flanking region of the gene by PCR. The 2318 bp genomic fragment contains the basal promoter of human CRLR, including potential TATA-boxes and several GC boxes. Regulatory elements binding known transcription factors, such as Sp-1, Pit-1, glucocorticoid receptor, and hypoxia-inducible factor-1 alpha (HIF-1alpha) were also identified. When cloned into reporter gene vectors, the genomic fragment showed significant promoter activity, indicating that the 5'-flanking region isolated by PCR contains the gene promoter of human CRLR. Of significance is that the cloned promoter fragments were activated by hypoxia when transfected in primary microvascular endothelial cells. Site-directed mutagenesis of the consensus hypoxia-response element (HRE) in the 5'-flanking region abolished such a response. We also demonstrated by semi-quantitative RT-PCR that transcription of the gene is activated by hypoxia in microvascular endothelial cells. In contrast, expression of RAMPs 1, 2, and 3 was unaffected by low oxygen tension. We conclude that simultaneous transcriptional up-regulation of CRLR and its ligand adrenomedullin in endothelial cells could lead to a potent survival loop and therefore might play a significant role in vascular responses to hypoxia and ischemia.

5' Flanking Region↗

PubMed1993

Targeting TBP to a non-TATA box cis-regulatory element: a TBP-containing complex activates transcription from snRNA promoters through the PSE.

In the human small nuclear RNA (snRNA) promoters, the presence of a TATA box recognized by the TATA box-binding protein (TBP) determines the selection of RNA polymerase III over RNA polymerase II. The RNA polymerase II snRNA promoters are, therefore, good candidates for TBP-independent promoters. We show here, however, that TBP activates transcription from RNA polymerase II snRNA promoters through a non-TATA box element, the snRNA proximal sequence element (PSE), as part of a new snRNA-activating protein complex (SNAPc). In contrast to the previously identified TBP-containing complexes SL1, TFIID, and TFIIIB, which appear dedicated to transcription by a single RNA polymerase, SNAPc is also essential for RNA polymerase III transcription from the U6 snRNA promoter. The U6 initiation complex appears to contain two forms of TBP, one bound to the TATA box and one bound to the PSE as a part of SNAPc, suggesting that multiple TBP molecules can have different functions within a single promoter.

Animals↗

PubMed1989

Negative and positive promoter elements contribute to tissue specificity of apolipoprotein B expression.

Apolipoprotein B (ApoB) is a major constituent of the plasma lipoproteins. In adult mammals it is synthesized in two different tissues, liver and intestine. We have examined the promoter elements involved in determining the cell specificity of apoB expression, using a chloramphenicol acetyltransferase assay and the cell lines HepG2, CaCo-2 and HeLa. The human apoB promoter contains: (i) a strong, cell-specific, positive element which can act on a heterologous promoter. This element is located between pos -111 and -33 and is built up by three subdomains, two positive and one negative; (ii) a large, negative element between pos -639 and -129, which reduces promoter activity in apoB expressing cells (HepG2 and CaCo-2) and block activation of the promoter by the SV40 enhancer in non-expressing cells (HeLa); (iii) a positive element in the noncoding part of exon 1 which retains its activity if placed upstream from the other regulatory elements and stimulates transcription from the simian virus 40 promoter in all three cell lines. The same sequence elements appear to be important for expression in cells of hepatic (HepG2) and intestinal (CaCo-2) origin.

Apolipoproteins B↗

PubMed1981

Genetic regulation: yeast mutants constitutive for beta-galactosidase activity have an increased level of beta-galactosidase messenger ribonucleic acid.

Mutants of Kluyveromyces lactis with elevated uninduced levels of beta-galactosidase (EC 32.1.2.3) activity, constitutive mutants (lac10c), were isolated and characterized to determine the basis for their constitutiveness. These lesions are not operator-type regulatory mutants because they are not closely linked to the beta-galactosidase structural gene. In a constitutive strain having a 7-fold increase in beta-galactosidase activity, the concentration of beta-galactosidase messenger ribonucleic acid (mRNA) was 8- to 10-fold higher than uninduced wild type. The half-life of beta-galactosidase mRNA was the same in the mutant strain (t1/2 = 4.5 +/- 0.2 min) as in uninduced wild-type cells (t1/2 = 4.8 +/- 0.1 min), indicating that the elevated mRNA level in the mutant was not due to a decreased rate of mRNA degradation. Consequently, we hypothesize that the LAC10 product regulates transcription of the beta-galactosidase gene; it probably affects the rate of transcription initiation. Parallel increases in enzyme protein, in constitutive levels of beta-galactosidase activity, and in mRNA further support this position, making translational or posttranslational control by LAC10 unlikely. Several types of data suggest that the LAC10 product functions as a negative regulatory element to prevent transcription. Other data demonstrate that lac10c mutations have pleiotrophic effects, there being constitutive levels not only of beta-galactosidase activity, but also the other lactose-inducible activities of galactokinase (EC 2.7.5.1), galactose-1-phosphate uridyl transferase (EC 2.7.7.10), and lactose transport. It would appear that LAC10 regulates lactose-inducible proteins.

Chromosome Mapping↗

PubMed2002

Identification of Egr1 as the oncostatin M-induced transcription activator that binds to sterol-independent regulatory element of human LDL receptor promoter.

Previously, we identified the low density lipoprotein receptor (LDLR) promoter region -17 to -1 as a novel sterol-independent regulatory element (SIRE) that mediates the stimulating effect of oncostatin M (OM). The goal of this study was to identify the OM-induced transcription activator that binds to the SIRE sequence. By conducting a electrophoretic mobility shift assay (EMSA) followed by UV crosslinking and SDS-PAGE, we show that a protein with a molecular mass of 85 kDa was present in the OM-induced SIRE DNA-protein complex. Western blotting and supershift assays reveal that the 85 kDa factor is early growth response gene 1 (Egr1). The interaction of Egr1 with the SIRE sequence was further confirmed in vivo by chromatin immunoprecipitation assays. The functional role of Egr1 in LDLR transcription was assessed by cotransfection of an Egr1 expression vector with an LDLR promoter reporter construct. We show that overexpression of Egr1 significantly increases LDLR promoter activity when cotransfected with CCAAT/enhancer binding protein beta (c/EBPbeta) or with cAMP-responsive element binding protein (CREB) expression vectors. Our studies clearly demonstrate that Egr1 is the OM-induced transcription factor that binds to the SIRE sequence of the LDLR promoter and also suggest that Egr1 may have a functional role in OM-induced upregulation of LDLR transcription through interaction with other SIRE binding proteins such as c/EBPbeta or CREB.

DNA-Binding Proteins↗

PubMedDate not supplied

Comparative analysis on the structural features of the 5' flanking region of kappa-casein genes from six different species.

Kappa-casein plays an essential role in the formation, stabilisation and aggregation of milk micelles. Control of kappa-casein expression reflects this essential role, although an understanding of the mechanisms involved lags behind that of the other milk protein genes. We determined the 5'-flanking sequences for the murine, rabbit and human kappa-casein genes and compared them to the published ruminant sequences. The most conserved region was not the proximal promoter region but an approximately 400 bp long region centred 800 bp upstream of the TATA box. This region contained two highly conserved MGF/STAT5 sites with common spacing relative to each other. In this region, six conserved short stretches of similarity were also found which did not correspond to known transcription factor consensus sites. On the contrary to ruminant and human 5' regulatory sequences, the rabbit and murine 5'-flanking regions did not harbour any kind of repetitive elements. We generated a phylogenetic tree of the six species based on multiple alignment of the kappa-casein sequences. This study identified conserved candidate transcriptional regulatory elements within the kappa-casein gene promoter.

5' Flanking Region↗

PubMed2004

Up-regulation of p21 gene expression by peroxisome proliferator-activated receptor gamma in human lung carcinoma cells.

PURPOSE: The peroxisome proliferator-activated receptor gamma (PPARgamma), a ligand-dependent transcription factor belonging to the family of nuclear receptors, has been implicated in the regulation of cell growth and differentiation although the exact mechanism(s) of this activity has not been elucidated. In this study, we explored the role of PPARgamma signaling on the control of gene expression of the cycle-dependent kinase inhibitor p21 in human lung carcinoma cells. EXPERIMENTAL DESIGN: Using several human lung carcinoma cell lines (small and non-small carcinoma cells), we assayed for cell growth inhibition and apoptosis induction. We also assayed for p21 mRNA and protein expression by reverse transcription-PCR, real-time reverse transcription-PCR, and Western blot analysis. Nuclear protein binding activities to three response elements located in the p21 promoter [nuclear factor (NF)-kappaB, Sp1, and NF-interleukin 6 (IL6) CAAT/enhancer binding protein (C/EBP)] were measured by gel mobility shift assays. We used transient transfection assays with p21 promoter reporter gene constructs to determine the transcriptional regulation by PPARgamma ligands. Finally, by using p21 antisense oligonucleotides, we tested the link between PPARgamma activation and p21 signaling in cell growth inhibition assays and by Western blot analysis. RESULTS: We showed that the PPARgamma ligands PGJ2 and ciglitazone inhibit the growth and induce the apoptosis of several human lung carcinoma cell lines, whereas the PPARalpha agonist WY14643 has little effect. Treatment of lung carcinoma cells with the PPARgamma ligands PGJ2, ciglitazone, troglizaone, and GW1929 elevated p21 mRNA and protein levels and reduced cyclin D1 mRNA levels. These results were supported by transient transfection assays, which indicated that PPARgamma ligands increased p21 gene promoter activity in human lung carcinoma cells. In addition, p21 antisense oligonucleotides inhibited PPARgamma ligand-induced p21 protein expression and significantly blocked lung carcinoma cell growth inhibition induced by PPARgamma ligands. Finally, electrophoresis mobility shift experiments demonstrated that PPARgamma ligands increased the nuclear binding activities of Sp1 and NF-IL6 (C/EBP), two transcription factors with regulatory elements in the promoter region of the p21 gene. CONCLUSION: PPARgamma ligands inhibit human lung carcinoma cell growth and induce apoptosis by stimulating the cyclin-dependent kinase inhibitor p21 and by reducing cyclin D1 gene expression. The induction of p21 gene expression by PPARgamma ligands may be mediated through increased Sp1- and NF-IL6 (C/EBP)-dependent transcriptional activation. These observations unveil a mechanism for p21 gene regulation in lung carcinoma that represents a potential target for therapy.

Apoptosis↗

PubMed1998

Differential regulation of basal and cyclic adenosine 3',5'-monophosphate-induced somatostatin gene transcription in neural cells by DNA control elements that bind homeodomain proteins.

A number of genes encoding neuropeptides are expressed in the peripheral and central nervous systems, in different endocrine organs, and in specialized cells distributed along the gastrointestinal tract. Whether expression of the same neuropeptide gene in different tissues is regulated by similar transcriptional mechanisms or by mechanisms that differ in a cell-specific manner remains unclear. We report on promoter studies on the regulation of the somatostatin gene in immortalized neural precursor cells derived from developing rat forebrain. Expression of the somatostatin gene in these cells was determined by RT-PCR/Southern blot analysis, by immunocytochemistry, and by RIA. We show that in cerebrocortical and hippocampal cells, expression of the somatostatin gene is regulated by several negative and positive DNA cis-regulatory elements located throughout the promoter region. The somatostatin cAMP-response element appears to play a prominent role in neural somatostatin gene expression by acting as a strong enhancer even in the absence of cAMP stimulation. Site-directed mutagenesis followed by transient transfection assays indicated that SMS-TAAT1, SMS-TAAT2, and SMS-UE, three previously identified homeodomain protein-binding regulatory elements that enhance transcription in pancreatic cells, act as repressors of transcription in neural cells. Electrophoretic mobility shifts assays indicate that those elements bind protein complexes that differ between neural and pancreatic cells. Our results support the notion that expression of the somatostatin gene in neural cells occurs via transcriptional mechanisms that are different from those regulating expression of the same gene in pancreatic cells.

Animals↗