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Results for “regulatory element transcripts”

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PubMed2005

Modulation of coreceptor transcription during positive selection dictates lineage fate independently of TCR/coreceptor specificity.

For developing T cells, coreceptor choice is matched to T cell antigen receptor (TCR) MHC specificity during positive selection in the thymus, but the mechanism remains uncertain. Here, we document that TCR-mediated positive selection signals inactivate the immature CD8(III) enhancer in double positive (DP) thymocytes, explaining in part the cessation of CD8 coreceptor transcription that occurs during positive selection. More importantly, by placing CD4 protein expression under the control of CD8 transcriptional regulatory elements, we demonstrate that cessation of CD4 coreceptor transcription during positive selection results in precisely the same lineage fate as cessation of CD8 coreceptor transcription. That is, MHC-II-signaled DP thymocytes differentiated into CD8-lineage cytotoxic T cells, despite the MHC-II specificity and CD4 dependence of their TCRs. This study demonstrates that termination of coreceptor transcription during positive selection promotes CD8-lineage fate, regardless of TCR specificity or coreceptor protein identity.

Animals↗

PubMed2002

Design and function of transcriptional switches in Drosophila.

Extensive genetic and biochemical analysis of Drosophila melanogaster has made this system an important model for characterization of transcriptional regulatory elements and factors. Given the striking conservation of transcriptional controls in metazoans, general principles derived from studies of Drosophila are expected to continue to illuminate transcriptional regulation in other systems, including vertebrates. With improvement in technologies for genetic manipulation of insects, research in Drosophila will also aid the design of systems for controlled expression of genes in other hosts. This review focuses on recent advances from Drosophila in analysis of the functional components of transcriptional switches, including basal promoters, enhancers, boundary elements, and maintenance elements.

Animals↗

PubMed2002

LCR-mediated, long-term tissue-specific gene expression within replicating episomal plasmid and cosmid vectors.

Locus control regions (LCRs) are transcriptional regulatory elements, which possess a dominant chromatin remodelling and transcriptional activating capability conferring full physiological levels of expression on a gene linked in cis, when integrated into the host cell genome. Using the human beta-globin LCR (betaLCR) as a model, we show that this class of control element can drive high levels of tissue-specific gene expression in stably transfected cultured cells from within an Epstein-Barr virus-based plasmid REV. Furthermore, a 38-kb betaLCR minilocus-REV cosmid vector was efficiently retained and maintained therapeutic levels of beta-globin transgene expression in the absence of drug selective pressure over a 2-month period of continuous culture equivalent to at least 60 generations. This demonstrates for the first time the feasibility of using REVs for gene therapy of the haemoglobinopathies. Importantly, our results demonstrate that as in the case of integrated transgenes, expression from within REVs is prone to silencing but that the inclusion of the betaLCR prevented this repression of gene function. Therefore, appropriate control elements to provide and maintain tissue-specific gene expression, as well as the episomal status of REVs is a crucial feature in vector design. Our data suggest that LCRs can contribute to this vital function.

Cell Line↗

PubMed1999

High level transcription of the complement regulatory protein CD59 requires an enhancer located in intron 1.

CD59 is a complement regulatory protein and may also act as a signal-transducing molecule. CD59 transgenic mice have been generated using a CD59 minigene (CD59 minigene-1). Although this minigene contained a 4.6-kilobase pair 5'-flanking region from the human CD59 gene as a promoter, the expression levels of the CD59 mRNA were substantially lower than those observed in humans, suggesting that CD59 gene expression might also require other transcriptional regulatory elements such as an enhancer. To investigate the transcriptional regulation of the CD59 gene, we used three cell lines that express CD59 at different levels. We have identified DNase I-hypersensitive sites in intron 1 in HeLa cells, which express CD59 at high levels, but not in Jurkat (intermediate level) or Raji cells (low level). Furthermore, cell line-specific enhancer activity was detected in a fragment containing these DNase I-hypersensitive sites. The CD59 enhancer was mapped to between -1155 and -888 upstream of the 5'-end of exon 2. To investigate the enhancer activity in vivo, a new CD59 minigene was constructed by the addition of the enhancer fragment into CD59 minigene-1. High expressor CD59 transgenic mice were generated using the new minigene.

Animals↗

PubMed1989

Human dopamine beta-hydroxylase gene: two mRNA types having different 3'-terminal regions are produced through alternative polyadenylation.

Two kinds of cDNA (types A and B) encoding human dopamine beta-hydroxylase (DBH) were isolated from a pheochromocytoma cDNA library. Type A (2.7 kb) and B (2.4 kb) encoded the same amino acid sequence and were different only in 3'-untranslated region. Type A contained 3'-extension of 300 bp at the end of type B. Subsequently, we isolated human DBH gene and analyzed genomic DNA by Southern hybridization. Human DBH gene (approximately 23 kb) was composed of 12 exons and existed as a single gene on genome. Exon 12 encoded 3'-terminal region of 1,013 bp of type A, including the 300 bp sequence. These results indicate that alternative use of two polyadenylation sites from a single DBH gene generates different mRNA types. This conclusion was supported by Northern hybridization and S1 nuclease mapping experiments. The ratio of type A and B mRNAs in pheochromocytoma was roughly 1.0 to 0.2. We found possible transcription regulatory elements, TATA, CCAAT, CACCC, GC boxes, near the transcription initiation site of DBH gene. Sequences homologous to glucocorticoid and cyclic AMP response elements were also observed.

Amino Acid Sequence↗

PubMedDate not supplied

A nuclear factor that binds to a conserved sequence motif in transcriptional control elements of immunoglobulin genes.

Trans-acting factors that mediate B-cell specific transcription of immunoglobulin genes have been postulated based on an analysis of the expression of exogenously introduced immunoglobulin gene recombinants in lymphoid and non-lymphoid cells. Two B-cell-specific, cis-acting transcriptional regulatory elements have been identified. One element is located in the intron between the variable (V) and constant (C) regions of both heavy and kappa light-chain genes and acts as a transcriptional enhancer. The second element is found upstream of both heavy and kappa light-chain gene promoters. This element directs lymphoid-specific transcription even in the presence of viral enhancers. We have sought nuclear factors that might bind specifically to these two regulatory elements by application of a modified gel electrophoresis DNA binding assay. We report here the identification of a human B-cell nuclear factor (IgNF-A) that binds to DNA sequences in the upstream regions of both the mouse heavy and kappa light-chain gene promoters and also to the mouse heavy-chain gene enhancer. This sequence-specific binding is probably mediated by a highly conserved sequence motif, ATTTGCAT, present in all three transcriptional elements. Interestingly, a factor showing similar binding specificity to IgNF-A is also present in human HeLa cells.

B-Lymphocytes↗

PubMedDate not supplied

Pyruvate kinase regulatory element 1 (PKR-RE1) mediates hexokinase gene expression in K562 cells.

We have established the functional importance of PKR-RE1, a necessary transcriptional regulatory element in the erythroid-specific promoter of the human pyruvate kinase gene (PKLR). Here, we demonstrate by electrophoretic mobility shift assay (EMSA) that the DNA-protein interaction at PKR-RE1 involves a CTGTC motif. Because the same motif is also present in the erythroid-specific promoter of the hexokinase gene (HK1), we confirmed its functional relevance by in vitro transfection in K562 cells. Moreover, EMSA demonstrated that the CTGTC motif in both the PKLR and HK1 promoters mediates binding of the same protein. Therefore, we postulate a more general role of PKR-RE1 in erythroid-specific gene expression.

Base Sequence↗

PubMed1993

The mouse Eb meiotic recombination hotspot contains a tissue-specific transcriptional enhancer.

A meiotic recombination hotspot exists within the second intron of the mouse major histocompatibility complex (MHC) gene, Eb. In the present study, a small fragment from the intron which contains two potential transcriptional regulatory elements was cloned into an expression vector and its effect on transcription was tested. This fragment was found to contain tissue-specific transcriptional enhancer activity. An octamer-like sequence and a B motif may contribute to this enhancer activity. Similar regulatory sequences with the same orientation and distance from one another are found in another mouse MHC recombination hotspot.

Animals↗

PubMed1999

Characterization and purification of carbohydrate response element-binding protein of the rat L-type pyruvate kinase gene promoter.

The L-III transcriptional regulatory element of the rat pyruvate kinase L gene is located between -170 and -150 base pairs upstream of the hepatocyte-specific transcription initiation site. As the L-III element is not only necessary for cell type-specific expression but also for transcriptional stimulation by carbohydrates, it is also referred to as a carbohydrate-response element. Electrophoretic mobility shift assays using rat liver nuclear extract showed that L-III element-binding protein (L-IIIBP) was observed as multiple bands. These bands disappeared when the nuclear extract was preincubated at 60 degrees C for 5 min and were competed with unlabeled L-III oligonucleotide but not with unlabeled adenovirus major late promoter E box oligonucleotide. In addition, these bands were not affected in the presence of antiserum against upstream stimulating factor (USF). Thus, we conclude that L-IIIBP is different from USF. Then, heat-labile L-IIIBP was purified from rat liver nuclear extracts. Purified L-IIIBP exhibited two bands on sodium dodecyl sulfate/polyacrylamide gel electrophoresis by silver staining. Ultraviolet crosslinking experiment showed that both bands had binding activity to the L-III oligonucleotide.

Adenoviridae↗

PubMed2004

Transcription factor sterol regulatory element binding protein 2 regulates scavenger receptor Cla-1 gene expression.

OBJECTIVE: The human scavenger receptor class B type I (Cla-1) plays a key role in cellular cholesterol movement in facilitating transport of cholesterol between cells and lipoproteins. Indirect evidence has suggested that Cla-1 gene expression is under the feedback control of cellular cholesterol content. To define the molecular mechanisms underlying such putative regulation, we evaluated whether Cla-1 is a target gene of the sterol regulatory element binding protein (SREBP) transcription factor family. METHODS AND RESULTS: Transient transfections demonstrated that SREBP factors induce Cla-1 promoter activity and that SREBP-2 is a more potent inducer than the SREBP-1a isoform. The 5'-deletion analysis of 3 kb of the 5'-flanking sequence of the Cla-1 gene, combined with site-directed mutagenesis and electrophoretic mobility shift assay, allowed identification of a unique sterol responsive element. SREBP-mediated Cla-1 regulation was confirmed in stably transfected human embryonic kidney 293 cells expressing the active form of SREBP-2 at incremental levels. In these cell lines, Cla-1 mRNA and protein levels were increased in direct proportion to the level of SREBP-2 expression. CONCLUSIONS: These findings provide evidence that SREBP-2, a key regulator of cellular cholesterol uptake through modulation of the expression of the low-density lipoprotein receptor gene, may influence cellular cholesterol homeostasis via regulation of Cla-1 gene expression.

Binding Sites↗

PubMed2000

Characterization of the rat connexin40 promoter: two Sp1/Sp3 binding sites contribute to transcriptional activation.

OBJECTIVES: The gap junction protein connexin40 (Cx40) is differentially expressed during embryonic development and in adult tissues, for which the molecular basis is unknown. In order to elucidate the molecular mechanisms controlling Cx40 expression, we set out to map and characterize its promoter. METHODS: The transcriptional activity of individual rat Cx40 (rCx40)-derived promoter fragments fused to the luciferase reporter gene was determined by transfection/reporter assays in Cx40-expressing (A7r5, rat smooth muscle embryonic thoracic aorta cells, and BWEM, v-myc transformed rat fetal cardiomyocytes) and Cx40-nonexpressing cells (N2A, mouse neuroblastoma cells). The nature of DNA-protein interactions was investigated by a combination of standard electrophoretic-mobility-shift assays (EMSA) and EMSA/antibody supershift assays. RESULTS: Quantification of luciferase activity in cell lysates revealed that a 235-base-pair fragment, in between map positions -150 and +85 relative to the transcription initiation site, is able to provide for a significant level of transcription in both Cx40-expressing (A7r5, BWEM) and -nonexpressing (N2A) cells. These results indicate that this region contains the basal promoter but is not sufficient to completely determine the endogenous Cx40-expression pattern within these cell types. In search for the responsible transcriptional regulatory element(s), additional segments of the (-150, +85) region were deleted and the remaining fragments were tested for transcriptional activity. These studies established that the regions in between map positions (-96, -71) and (+58, +85) contribute to promoter activity. EMSA with these regions revealed that predominantly two DNA-protein complexes are formed upon incubation with either A7r5, BWEM or N2A nuclear extracts, which could be both inhibited by including excess oligonucleotide containing the Sp1 consensus binding site in the binding reaction. Purified recombinant human Sp1 provided also for a shift in the EMSA using these promoter regions as target fragments. When the DNA-protein complexes formed with nuclear extract were subsequently incubated with either an anti-Sp1 or an anti-Sp3 antibody clear supershifts in the EMSA were obtained, indicating Sp1 and Sp3 binding to both the (-98, -64) and (+53, +87) regions. The introduction of mutations within the core sequence of the putative Sp1/Sp3 binding sites present in these regulatory elements reduced the level of transcriptional activity and abrogated Sp1/Sp3 binding to these sites. CONCLUSION: The results indicate that at least two Sp1/Sp3 binding sites in the rCx40 promoter contribute to the transcriptional activation of its gene in cultured cells.

Animals↗

PubMed1998

The macrosialin promoter directs high levels of transcriptional activity in macrophages dependent on combinatorial interactions between PU.1 and c-Jun.

Macrosialin is a transmembrane glycoprotein that is highly expressed in macrophages. In the present studies, macrosialin mRNA levels are shown to be markedly up-regulated during macrophage differentiation of bone marrow progenitor cells in response to macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. To investigate the mechanisms responsible for regulation of macrosialin expression, we have isolated the macrosialin gene and performed an initial analysis of its transcriptional regulatory elements. The macrosialin promoter and 7.0 kilobase pairs of 5'-flanking information direct high levels of reporter gene activity in monocyte/macrophage-like cells, but little or no expression in nonmyeloid cells. This pattern of expression is dependent on regulatory elements located between -7.0 and -2.5 kilobase pairs from the transcriptional start site that exhibit strong enhancer activity in macrophages and repressor activity in nonmyeloid cells. Analysis of the proximal macrosialin promoter indicates that combinatorial interactions between at least four classes of transcriptional activators, including PU.1/Spi-1 and members of the AP-1 family are required for basal promoter function. PU.1/Spi-1 and c-Jun act synergistically to activate the macrosialin promoter in a nonmyeloid cell line, suggesting that combinatorial interactions between these proteins are involved in regulating macrosialin expression during macrophage differentiation.

Amino Acid Sequence↗

PubMed1994

Isolation of genomic DNA fragments corresponding to genes modulated in vivo by a transcription factor.

A new methodology for the identification of genes modulated by transcription factors in vivo is described. Mouse genomic DNA fragments bound by the thyroid hormone receptor (T3R) were selected and amplified in vitro. Subsequent hybridisation with biotinylated cDNA allowed the selection of those DNA fragments containing binding sites for T3R that corresponded to transcribed DNA. Expression analysis of the corresponding genes showed that more than 80% are indeed modulated by thyroid hormones in vivo in the liver. Together with the presence of consensus binding sites for T3R this result suggests that the selected DNA fragments may contain T3R transcriptional regulatory elements. This method, extensive to other ligand-modulated transcription factors, might be useful to all transcription factors with slight modifications.

Animals↗

PubMed2005

STAT4 expression in human T cells is regulated by DNA methylation but not by promoter polymorphism.

STAT4, which plays a pivotal role in Th1 immune responses, enhances IFN-gamma transcription in response to the interaction of IL-12 with the IL-12R. Mice deficient in STAT4 lack IL-12-induced IFN-gamma production and Th1 differentiation and display a predominantly Th2 phenotype. Although these findings indicate that STAT4 expression levels are important for the development of cytokine-producing Th1 cells, the transcriptional and posttranscriptional mechanisms regulating STAT4 expression are unknown. We sought to identify and characterize the transcriptional regulatory elements in the promoter region of the human STAT4 gene. We found that disruption of multiple transcriptional regions covering the CREB, OCT1, and SP1 motifs significantly reduced STAT4 promoter activity. However, genomic DNA isolated from 91 patients with asthma or rheumatoid arthritis showed no evidence of mutations in the defined STAT4 essential promoter region. The 5' flanking region of the promoter was found to contain a -149A/G change in approximately 20-35% of patients, but this polymorphism had no effect on promoter activity. Interestingly, STAT4 expression was drastically increased in human T cells following treatment with a DNA methyltransferase inhibitor, and truncation of methylation sites in the proximal regulatory elements of the STAT4 promoter markedly enhanced transcriptional activity. Thus, our findings provide molecular insight into STAT4 expression and suggest that, in human T cells, STAT4 expressional regulation is associated with DNA hypermethylation, but not promoter polymorphisms.

Asthma↗

PubMed1994

Identification of a TPA-responsive element mediating preferential transactivation of the galanin gene promoter in chromaffin cells.

The gene encoding the neuropeptide galanin is upregulated by second messenger signal transduction pathways in bovine chromaffin cells. To identify its transcriptional regulatory elements, 5'-flanking sequences of the galanin gene were transiently transfected into primary cultures of bovine chromaffin cells within reporter gene constructs. Multiple regions of the galanin 5' flank seem to be necessary for basal activity. The most promoter-proximal of these regions contains a sequence (TGACG) -66 to -62 nucleotides upstream from the transcriptional start site which mediates stimulation by 12-O-tetradecanoylphorbol-13-acetate (TPA), as demonstrated by site-directed mutagenesis and cis-activation experiments. This cis-regulatory element mediates preferential TPA stimulation of transcription from the galanin promoter in chromaffin cells compared with bovine endothelial or HeLa cells. DNA-protein binding assays indicate that an oligonucleotide that includes the galanin TPA-responsive element (GTRE) binds specifically to proteins from nuclear extracts of chromaffin cells. TPA treatment persistently increases this binding activity in chromaffin but not in endothelial cells. Mutation of the galanin promoter within the -66 to -62 region renders it unresponsive to transcriptional stimulation by TPA, and a correspondingly mutated oligonucleotide fails to bind chromaffin cell nuclear proteins in a gel-shift assay. Chromaffin cell nuclear extracts also contain proteins that bind consensus TPA-responsive (TRE) and cyclic AMP-responsive (CRE) elements. GTRE, TRE, and CRE oligonucleotides all compete more efficiently for protein binding to their labeled congeners than for protein binding to either of the other labeled oligonucleotides, suggesting that the GTRE, TRE, and CRE oligonucleotides, suggesting that the GTRE, TRE, and CRE oligonucleotides each bind unique as well as common proteins, likely to be members of the Jun/Fos and cAMP-responsive element-binding protein/activating transcription factors (CREB/ATF) families of transcription factors, in chromaffin cells.

Adrenal Medulla↗

PubMed1994

Transcriptional regulation of the human Wilms' tumor gene (WT1). Cell type-specific enhancer and promiscuous promoter.

The Wilms' tumor gene, WT1, is expressed in few tissues, mainly the developing kidney, genitourinary system, and mesothelium, and in immature hematopoietic cells. To develop an understanding of the role of WT1 in development and tumorigenesis, we have identified transcriptional regulatory elements that function in transient reporter gene constructs transfected into kidney and hematopoietic cell lines. We found three transcription start sites of the WT1 gene and have identified an essential promoter region by deletion analysis. The WT1 promoter is a member of the GC-rich, TATA-less, and CCAAT-less class of polymerase II promoters. Whereas the WT1 promoter is similar to other tumor suppressor gene promoters, the WT1 expression pattern (unlike Rb and p53) is tissue-restricted. The WT1 GC-rich promoter is promiscuous, functioning in all cell lines tested, independent of WT1 expression. This finding suggests that the promoter is not tissue-specific, but that tissue-specific expression of WT1 is modulated by additional regulatory elements. Indeed, we have identified a transcriptional enhancer located 3' of the WT1 gene > 50 kilobases downstream from the promoter. This orientation-independent enhancer increases the basal transcription rate of the WT1 promoter in the human erythroleukemia cell line K562, but not in any of the other cell lines tested.

Base Sequence↗

PubMed1998

Divergent transcriptional control of multidrug resistance genes in Saccharomyces cerevisiae.

Improper control of expression of ATP binding cassette transporter-encoding genes is an important contributor to acquisition of multidrug resistance in human tumor cells. In this study, we have analyzed the function of the promoter region of the Saccharomyces cerevisiae YOR1 gene, which encodes an ATP binding cassette transporter protein that is required for multidrug tolerance in S. cerevisiae. Deletion analysis of a YOR1-lacZ fusion gene defines three important transcriptional regulatory elements. Two of these elements serve to positively regulate expression of YOR1, and the third element is a negative regulatory site. One positive element corresponds to a Pdr1p/Pdr3p response element, a site required for transcriptional control by the homologous zinc finger transcription factors Pdr1p and Pdr3p in other promoters. The second positive element is located between nucleotides -535 and -299 and is referred to as UASYOR1 (where UAS is upstream activation sequence). Interestingly, function of UASYOR1 is inhibited by the downstream negative regulatory site. Promoter fusions constructed between UASYOR1 and the PDR5 promoter, another gene under Pdr1p/Pdr3p control, are active, whereas analogous promoter fusions constructed with the CYC1 promoter are not. This suggests the possibility that UASYOR1 has promoter-specific sequence requirements that are satisfied by another Pdr1p/Pdr3p-regulated gene but not by a heterologous promoter.

ATP-Binding Cassette Transporters↗

PubMed2004

Intravenous administration of an AAV-2 vector for the expression of factor IX in mice and a dog model of hemophilia B.

Previous experiments have demonstrated the stable expression of factor IX (FIX) protein in mice and canine models of hemophilia B following portal vein gene transfer with a recombinant adeno-associated virus (rAAV) vector encoding FIX. Here, we present the results of studies that further optimized the rAAV vector transgene cassette used to express FIX and explored the use of the less-invasive intravenous (i.v.) route of vector administration for the treatment of hemophilia B. First, a liver-specific promoter was evaluated in conjunction with cis-acting regulatory elements in mice. Constructs that included both the beta-globin intron and the woodchuck hepatitis virus post-transcriptional regulatory element resulted in the highest level of FIX expression in vivo. Using this optimized vector, we demonstrate that i.v. injection was feasible for hepatic gene transfer in mice, achieving 70-80% of portal vein expression levels of FIX. In further studies using the Chapel Hill strain of hemophilia B dogs, we demonstrate for the first time FIX expression and partial correction of the bleeding disorder following i.v. administration of an AAV vector.

Animals↗