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Results for “regulatory element transcripts”

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PubMed2002

Recombinant adeno-associated virus vector design and gene expression in the mammalian brain.

Efficiency and stability of recombinant adeno-associated virus (rAAV)-mediated gene expression within the mammalian brain are determined by several factors. These include the dose of infectious particles, the purity of the vector stock, the serotype of rAAV, the route of administration, and the intrinsic properties, most notably the rAAV receptor density, of the targeted area. Furthermore, the choice of appropriate regulatory elements in rAAV vector design is of fundamental importance to achieve high-level sustained in vivo transcription and translation. This review summarizes the characteristics of various transcriptional and posttranscriptional regulatory elements, and highlights their influence on the expression performance of rAAV vectors in the mammalian brain.

Adenoviridae↗

PubMed1994

The transcription factor YY1 binds to negative regulatory elements in the human cytomegalovirus major immediate early enhancer/promoter and mediates repression in non-permissive cells.

We have previously shown that repression of human cytomegalovirus (HCMV) major immediate early (IE) gene expression in non-permissive human teratocarcinoma (T2) cells is associated with a number of nuclear factors which bind to the imperfect dyad symmetry located in the modulator region upstream of the major IE enhancer as well as to the 21 bp repeat elements within the enhancer. Differentiation of T2 cells with retinoic acid (RA) results in a decrease in binding of some of these nuclear factors to these sites and deletion of these specific binding sites from major IE promoter/reporter constructs results in increased IE promoter activity in normally non-permissive cells. In this study, we demonstrate that the transcription factor YY1, which can negatively regulate the adeno-associated virus P5 promoter, directly binds to both the imperfect dyad symmetry and the 21 bp repeat elements in the HCMV major IE promoter/regulatory region and mediates repression of HCMV IE gene expression. This strongly suggests that YY1 plays an important role in regulating HCMV expression in non-permissive cells.

Antigens, Viral↗

PubMed2002

Regulation of gene promoters of hypothalamic peptides.

In order to fulfill their roles in neuroendocrine regulation, specific hypothalamic neurons are devoted to produce and deliver biologically active peptides to the pituitary gland. The biosynthesis and release of peptides are strictly controlled by afferents to these hypothalamic neurons. Cell-specific expression and biosynthetic regulation largely relies on transcription from the gene promoter for which the 5(')-flanking regions of the peptidergic genes contain essential elements. Cell-specific transcription factors employ these regulatory elements to exert their control over the expression of the peptidergic gene. This article explores the properties of regulatory elements of the major hypothalamic peptides, somatostatin, growth hormone-releasing hormone, gonadotropin-releasing hormone, thyrotropin-releasing hormone, corticotropin-releasing hormone, vasopressin and oxytocin, and the transcription factors acting on them. These transcription factors are often endpoints of signal transduction pathways that can be activated by neurotransmitters or steroid hormones. Others are essential to provide cell-specific expression of the peptidergic gene during development and mature regulation.

Animals↗

PubMed2004

GH and insulin affect fatty acid synthase activity in isolated porcine adipocytes in culture without any modifications of sterol regulatory element binding protein-1 expression.

The ability of GH to decrease fatness and insulin-regulated events such as lipogenic enzyme activities is well known in pigs. Nevertheless, the precise mechanism underlying these actions has not been elucidated yet. Expression of the transcription factor sterol regulatory element binding protein (SREBP)-1 has been reported as a key mediator of insulin action in rat hepatocytes and adipose cell lines. The present study aimed to determine whether the regulation of lipogenesis by GH and/or insulin in porcine adipocytes also involved SREBP-1. Isolated adipocytes, obtained from perirenal or s.c. adipose tissue samples of female pigs (51+/-0.4 kg; n=17), were cultured in serum-free medium in the absence or presence of these hormones for up to 4 days. Glucose incorporation and fatty acid synthase activity were increased by insulin in a dose-dependent manner in adipocytes of both sites. The increase was maximal at 1.7 and 17 nM in s.c. and perirenal adipocytes respectively, suggesting inter-depot differences in the regulation of lipogenesis by insulin. These insulin-stimulated events were decreased by GH (1 nM). No change in SREBP-1 mRNA levels was observed in response to GH and/or insulin. Taken together, these data indicate that the regulation of lipogenesis by insulin and GH appears to not involve changes in SREBP-1 mRNA levels in porcine adipocytes.

Adipocytes↗

PubMed1992

Interaction of the erythroid transcription factor cGATA-1 with a critical auto-regulatory element.

We have performed a mutational analysis of the promoter for the chicken erythroid-specific GATA-1 transcription factor, and have investigated in detail the interaction of the factor with an upstream auto-regulatory element (ARE). We find that a single proximal GATA binding site of the ARE is required for promoter activity in primary erythroid cells; however, this minimal promoter is inappropriately active in fibroblasts. At least two molecules of GATA-1 can interact with the ARE, and sequences outside of the consensus site appear critical for the transcriptional activity of the bound protein. Finally, we provide evidence for complex protein/DNA interactions at the ARE, including the ability of GATA-1 to bend DNA.

Animals↗

PubMed1999

Redundant enhancers mediate transcriptional repression of AGAMOUS by APETALA2.

The floral homeotic gene AGAMOUS specifies stamen and carpel fate in the central whorls of Arabidopsis flowers. Transcription of AGAMOUS RNA is restricted to the center of developing flowers by several, partially redundant negative regulators, one of which is the homeotic gene APETALA2. We have identified regulatory elements that mediate transcriptional repression of AGAMOUS by APETALA2 and found that several redundant elements respond independently to loss of APETALA2 activity. Thus, redundancy at the level of cis-regulatory sequences is independent of redundancy at the level of trans-regulators. We have also found that only the early, but not the late, effects of APETALA2 on AGAMOUS require the meristem-identity protein LEAFY, a positive regulator of AGAMOUS.

AGAMOUS Protein, Arabidopsis↗

PubMed1993

Developmental regulation and butyrate-inducible transcription of the Xenopus histone H1(0) promoter.

We have isolated genomic clones of the Xenopus laevis histone H1(0) promoter and identified regulatory elements mediating the transcriptional regulation of the H1(0) gene. Expression of H1(0) is associated with the terminal differentiation of many cell types. During X. laevis development, H1(0) mRNA is present in the oocyte and egg, but remains at low levels during embryogenesis until hatching. After this time, mRNA levels accumulate dramatically correlating with the differentiation of many tissue types, e.g., liver and skin. Accumulation of H1(0) mRNA can be induced at earlier development stages by treating embryos with butyrate. The enhanced transcription of H1(0) in adult somatic cells, as well as the butyrate inducibility of the gene, have been investigated using transfection of adult X. laevis A6 somatic cells. We have defined specific protein-nucleic acid interactions with three cis-acting elements. Two previously defined gene regulatory elements: the H1 box, normally involved in the regulation of the H1 gene, and the H4TF2 site, normally involved in the regulation of the H4 gene, appear to have novel roles in determining differentiation-specific H1(0) expression. These two elements act together with a new distal cis-acting element in order to sustain high levels of basal transcription and to potentiate transcription following butyrate treatment.

Amino Acid Sequence↗

PubMed2000

Identification and functional characterization of the human and murine fibroblast growth factor receptor 4 promoters.

Fibroblast growth factor receptors (FGFRs) play crucial roles in signal transduction of adult tissues and during embryonic development. To study the transcriptional control, we isolated and characterized the promoter of human FGFR4. Two transcription initiation sites were identified. The deletion analysis in different cell types defined a core promoter reaching from -9 to -198, lacking TATA and CCAAT boxes but displaying high GC content (77%) in a stretch of 300 bp upstream of the major mRNA start. This region harbors multiple binding motifs for transcription factors. Moreover, the region between -1085 and -1140 contains a potential repressor element, which downregulates transcriptional activity. To identify conserved regulatory elements, we isolated and analyzed also the murine FGFR4 promoter. Only one transcription start was identified using RNase protection assays. Sequence alignment of human and mouse shows a striking similarity in the core promoter region of both genes, encompassing conserved transcription factor binding sites and a splice acceptor site. Furthermore, the region containing the putative repressor element is also conserved suggesting a functional role for gene expression.

Animals↗

PubMed2004

Interpreting mammalian evolution using Fugu genome comparisons.

Recently, it has been shown that a significant number of evolutionarily conserved human-Fugu noncoding elements function as tissue-specific transcriptional enhancers in vivo, suggesting that distant comparisons are capable of identifying a particular class of regulatory elements. We therefore hypothesized that by juxtaposing human/Fugu and human/mouse conservation patterns we can define conservation criteria for discovering transcriptional regulatory elements specific to mammals. Genome-scale comparisons of noncoding human/Fugu evolutionary conserved elements (ECRs) and their humans/mouse counterparts revealed a particular signature common to human/mouse ECRs (>or=350 bp long, >or=77% identity) that are also conserved in fishes. This newly defined threshold identifies 90% of all human/Fugu noncoding ECRs without the assistance of human-Fugu genome alignments and provides a very efficient filter for identifying functional human/mouse ECRs.

Animals↗

PubMed1992

Mechanism of tissue-specific transcription: interplay between positive and negative regulatory factors.

At least four regulatory cis-acting DNA sequences, CCAAAAGTGG (element A), TTATTTTTA (element B), TATTTATT (element C), and TATTACCTTTAT (element S), were identified in cardiac myosin light chain-2 (MLC2) proximal promoter as target sites for sequence-specific binding of nuclear proteins. For muscle-specific transcription, the proximal promoter (-53 to +1) consisting only of elements B and C is required. Addition of element A to this promoter results in a muscle-specific up-regulation, whereas the addition of element S exerts a negative effect on transcription. The negative and positive regulatory effects of elements S and A respectively were demonstrated by site-specific mutations of the promoter following transient transfection of cardiac muscle cells in culture. Elements S and A interact separately with distinct nuclear protein factor present in both muscle and non-muscle cells, even though their regulatory activities are restricted to muscle cells. Among the multiple complexes resulting from the interaction of nuclear proteins and elements S and A DNAs, one requires both S and A sequences together for binding. Element B, which exerts a muscle-specific positive effect on transcription, binds to a nuclear protein present in cardiac muscle, but not in non-muscle cells. DNA-protein binding assays and mutational analysis of the MLC2 promoter suggest that the contribution of the functionally opposed cis-elements depends upon an interplay between the positively and negatively acting DNA-binding proteins via protein-protein interactions to mediate opposite regulatory effects on gene transcription.

Animals↗

PubMed2006

Analysis of folylpoly-gamma-glutamate synthetase gene expression in human B-precursor ALL and T-lineage ALL cells.

BACKGROUND: Expression of folylpoly-gamma-glutamate synthetase (FPGS) gene is two- to three-fold higher in B-precursor ALL (Bp- ALL) than in T-lineage ALL (T-ALL) and correlates with intracellular accumulation of methotrexate (MTX) polyglutamates and lymphoblast sensitivity to MTX. In this report, we investigated the molecular regulatory mechanisms directing FPGS gene expression in Bp-ALL and T-ALL cells. METHODS: To determine FPGS transcription rate in Bp-ALL and T-ALL we used nuclear run-on assays. 5'-RACE was used to uncover potential regulatory regions involved in the lineage differences. We developed a luciferase reporter gene assay to investigate FPGS promoter/enhancer activity. To further characterize the FPGS proximal promoter, we determined the role of the putative transcription binding sites NFY and E-box on FPGS expression using luciferase reporter gene assays with substitution mutants and EMSA. RESULTS: FPGS transcription initiation rate was 1.6-fold higher in NALM6 vs. CCRF-CEM cells indicating that differences in transcription rate led to the observed lineage differences in FPGS expression between Bp-ALL and T-ALL blasts. Two major transcripts encoding the mitochondrial/cytosolic and cytosolic isoforms were detected in Bp-ALL (NALM6 and REH) whereas in T-ALL (CCRF-CEM) cells only the mitochondrial/cytosolic transcript was detected. In all DNA fragments examined for promoter/enhancer activity, we measured significantly lower luciferase activity in NALM6 vs. CCRF-CEM cells, suggesting the need for additional yet unidentified regulatory elements in Bp-ALL. Finally, we determined that the putative transcription factor binding site NFY, but not E-box, plays a role in FPGS transcription in both Bp- and T-lineage. CONCLUSION: We demonstrated that the minimal FPGS promoter region previously described in CCRF-CEM is not sufficient to effectively drive FPGS transcription in NALM6 cells, suggesting that different regulatory elements are required for FPGS gene expression in Bp-cells. Our data indicate that the control of FPGS expression in human hematopoietic cells is complex and involves lineage-specific differences in regulatory elements, transcription initiation rates, and mRNA processing. Understanding the lineage-specific mechanisms of FPGS expression should lead to improved therapeutic strategies aimed at overcoming MTX resistance or inducing apoptosis in leukemic cells.

Binding Sites↗

PubMed2005

A novel functional interaction between the Sp1-like protein KLF13 and SREBP-Sp1 activation complex underlies regulation of low density lipoprotein receptor promoter function.

Cholesterol homeostasis is regulated by a family of transcription factors designated sterol regulatory element-binding proteins (SREBPs). Precise control of SREBP-targeted genes requires additional interactions with co-regulatory transcription factors. In the case of the low density lipoprotein receptor (LDLR), SREBP cooperates with the specificity protein Sp1 to activate the promoter. In this report, we describe a novel pathway in LDLR transcriptional regulation distinct from the SREBP-Sp1 activation complex involving the Sp1-like protein Krueppel-like factor 13 (KLF13). Using a combination of RNA interference, electrophoretic mobility shift, chromatin immunoprecipitation, and reporter assays, deletion, and site-directed mutagenesis, we demonstrated that KLF13 mediates repression in a DNA context-selective manner. KLF13 repression of LDLR promoter activity appears to be needed to keep the receptor silent, a state that can be antagonized by Sp1, SREBP, and inhibitors of histone deacetylase activity. Chromatin immunoprecipitation assay confirmed that KLF13 binds proximal LDLR DNA sequences in vivo and that exogenous oxysterol up-regulates such binding. Together these studies identify a novel regulatory pathway in which gene repression by KLF13 must be overcome by the Sp1-SREBP complex to activate the LDLR promoter. Therefore, these data should replace a pre-existent and more simple paradigm that takes into consideration only the induction of the activator proteins Sp1-SREBP as necessary for LDLR promoter drive without including default repression, such as that by KLF13, of the LDLR gene.

Animals↗

PubMed2000

Early expression of herpes simplex virus (HSV) proteins and reactivation of latent infection.

During the last decade, new data accumulated describing the early events during herpes simplex virus 1 (HSV-1) replication occurring before capsid formation and virion envelopment. The HSV virion carries its own specific transcription initiation factor (alpha-TIF), which functions together with other components of the cellular transcriptase complex to mediate virus-specific immediate early (IE) transcription. The virus-coded IE proteins are the transactivator and regulatory elements modulating early transcription and subsequent translation of nonstructural virus-coded proteins needed mainly for viral DNA synthesis and for the supply of corresponding nucleoside components. They also cooperate at the late transcription and translation of the virion (capsid, tegument and envelope) proteins. In addition, the transactivator IE proteins down-regulate their own transcription, while others facilitate viral mRNA processing or interfere with the presentation of newly synthesized virus antigens. Establishment of latency is closely related to the transcription of a separate category of transcripts, termed latency-associated (LAT). Formation of LATs occurs mainly in nondividing neurons which are metabolically less active and express lower levels of cellular transcription factors (nonpermissive cells). Expression of the stable non-spliced (2 kb), and especially of stable spliced (1.5 and 1.45 kb) LATs is a prerequisite for HSV reactivation. Different HSV genomes (from various HSV strains) do not undergo IE transcription at the same rate. Restricted IE transcription and the absence of viral DNA synthesis favors LAT formation and persistence of the silenced genome. Uneven levels of LAT expression and differences in the metabolic state of carrier neurons influence the reactivation competence. Under artificial or natural activation conditions, sufficient amounts of IE transactivator proteins and proteins promoting nucleoside metabolism are synthesized even in the absence of the viral alpha-TIF facilitating reactivation.

Animals↗

PubMed2005

Analysis of human UDP-glucose dehydrogenase gene promoter: identification of an Sp1 binding site crucial for the expression of the large transcript.

UDP-glucose dehydrogenase (UGDH) catalyzes the conversion of UDP-glucose to UDP-glucuronic acid, which is required in liver for the excretion of toxic compounds, and for the biosynthesis of complex carbohydrates, such as hyaluronan, in many cell types. Analysis of a human EST database, as well as the results of a 5'-RACE experiment, have revealed the presence of two transcription start sites approximately 160 bp apart in the human UGDH gene confirming previous Northern hybridization results. To delineate the regions in the UGDH promoter required for regulating the expression of the gene, in particular the synthesis of the large transcript, serial deletions of the 2.1-kb UGDH promoter region were constructed and their activities determined by the firefly luciferase reporter gene assay. Our results indicate that the region from nucleotide position -486 to -632 relative to the start of the small transcript contains positive regulatory elements that contribute to gene expression. Mithramycin A, an inhibitor of transcription factor Sp1, abrogates the promoter activity, suggesting the involvement of this specific protein in UGDH expression. By using site-directed mutagenesis, we analyzed the functional contribution of three putative Sp1 binding elements within this region. A mutation at position -564 demonstrated that this site serves as an enhancing element in both HepG2 and HeLa cells. The complex formation pattern revealed by an electrophoretic mobility shift assay as well as an anti-Sp1 antibody-mediated supershift assay confirmed the identity of this GC box as an Sp1 binding motif. Our results thus identify an alternative transcription start site on the UGDH promoter, and locate the cis-element that greatly enhances the basal transcriptional activity of UGDH gene.

Base Sequence↗

PubMed1999

Sterol regulatory element binding protein-1c is a major mediator of insulin action on the hepatic expression of glucokinase and lipogenesis-related genes.

Hepatic glucokinase plays a key role in glucose metabolism as underlined by the anomalies associated with glucokinase mutations and the consequences of tissue-specific knock-out. In the liver, glucokinase transcription is absolutely dependent on the presence of insulin. The cis-elements and trans-acting factors that mediate the insulin effect are presently unknown; this is also the case for most insulin-responsive genes. We have shown previously that the hepatic expression of the transcription factor sterol regulatory element binding protein-1c (SREBP-1c) is activated by insulin. We show here in primary cultures of hepatocytes that the adenovirus-mediated transduction of a dominant negative form of SREBP-1c inhibits the insulin effect on endogenous glucokinase expression. Conversely, in the absence of insulin, the adenovirus-mediated transduction of a dominant positive form of SREBP-1c overcomes the insulin dependency of glucokinase expression. Hepatic fatty acid synthase and Spot-14 are insulin/glucose-dependent genes. For this latter class of genes, the dominant positive form of SREBP-1c obviates the necessity for the presence of insulin, whereas glucose potentiates the effect of SREBP-1c on their expression. In addition, the insulin dependency of lipid accumulation in cultured hepatocytes is overcome by the dominant positive form of SREBP-1c. We propose that SREBP-1c is a major mediator of insulin action on hepatic gene expression and a key regulator of hepatic glucose/lipid metabolism.

Animals↗

PubMed2005

mSin3A corepressor regulates diverse transcriptional networks governing normal and neoplastic growth and survival.

mSin3A is a core component of a large multiprotein corepressor complex with associated histone deacetylase (HDAC) enzymatic activity. Physical interactions of mSin3A with many sequence-specific transcription factors has linked the mSin3A corepressor complex to the regulation of diverse signaling pathways and associated biological processes. To dissect the complex nature of mSin3A's actions, we monitored the impact of conditional mSin3A deletion on the developmental, cell biological, and transcriptional levels. mSin3A was shown to play an essential role in early embryonic development and in the proliferation and survival of primary, immortalized, and transformed cells. Genetic and biochemical analyses established a role for mSin3A/HDAC in p53 deacetylation and activation, although genetic deletion of p53 was not sufficient to attenuate the mSin3A null cell lethal phenotype. Consistent with mSin3A's broad biological activities beyond regulation of the p53 pathway, time-course gene expression profiling following mSin3A deletion revealed deregulation of genes involved in cell cycle regulation, DNA replication, DNA repair, apoptosis, chromatin modifications, and mitochondrial metabolism. Computational analysis of the mSin3A transcriptome using a knowledge-based database revealed several nodal points through which mSin3A influences gene expression, including the Myc-Mad, E2F, and p53 transcriptional networks. Further validation of these nodes derived from in silico promoter analysis showing enrichment for Myc-Mad, E2F, and p53 cis-regulatory elements in regulatory regions of up-regulated genes following mSin3A depletion. Significantly, in silico promoter analyses also revealed specific cis-regulatory elements binding the transcriptional activator Stat and the ISWI ATP-dependent nucleosome remodeling factor Falz, thereby expanding further the mSin3A network of regulatory factors. Together, these integrated genetic, biochemical, and computational studies demonstrate the involvement of mSin3A in the regulation of diverse pathways governing many aspects of normal and neoplastic growth and survival and provide an experimental framework for the analysis of essential genes with diverse biological functions.

Animals↗

PubMed1996

Ret 4, a positive acting rhodopsin regulatory element identified using a bovine retina in vitro transcription system.

Previous transgenic mouse studies demonstrated that the bovine rhodopsin sequence between -222 and +70 base pairs (bp) contains a minimal promoter, which is sufficient to direct photoreceptor cell-specific expression of a lacZ reporter gene. To more fully define the DNA regulatory elements and protein factors involved in regulating rhodopsin transcription, we have developed an in vitro transcription system derived from bovine retinal nuclear extracts. Retinal extracts, as compared to liver, HeLa, and Drosophila embryonic cell extracts, demonstrated preferential activity for the rhodopsin promoter. A template spanning the bovine rhodopsin upstream region from -590 to +15 bp showed significant activation relative to the basal activity seen with a TATA box containing -38 to +15 bp template. Deletion analysis indicated that the region between -85 and -38 bp contained significant positive regulatory activity. This activity was not observed with HeLa extracts, suggesting that it might be retina-specific. Systematic site-directed mutagenesis of the subregion from -64 to -38 bp indicated that sequences between -60 and -58 bp and between -48 and -40 bp harbor critical elements. The former sequence is part of the binding site for the retina-specific transcription factor Nrl, which has been implicated in rhodopsin regulation. Electrophoretic mobility shift assays showed that the latter sequence (-48 to -40 bp), and flanking DNA, designated Ret 4, is bound by both retina-specific and ubiquitously expressed protein factors. Shift assays with mutant oligomers further defined the putative recognition sequences for these protein factors. Together, our results suggest that multiple promoter elements and transcriptional factors are involved in regulating photoreceptor-specific rhodopsin transcription.

Animals↗

PubMed1996

In vitro binding of the tomato bZIP transcriptional activator VSF-1 to a regulatory element that controls xylem-specific gene expression.

The bean grp1.8 gene is specifically expressed in vascular tissue. Monomers and multimers of a 28 bp regulatory element of the grp1.8 promoter (vs-1) specifically activated both the -82 CaMV 35S and the -76/grp1.8 minimal promoters in vascular tissue of transgenic tobacco plants. vs-1 partially overlaps with a negative regulatory element in the grp1.8 promoter that is necessary for restriction of gene expression to vascular tissue. Nuclear extracts from tobacco and tomato cells contain a factor that binds to vs-1 in vitro. To study the molecular basis of xylem-specific expression mediated by the vs-1 promoter element, a gene was isolated from tomato encoding a protein that binds to vs-1 in vitro. This protein, designated VSF-1, contains a bZIP motif close to the C-terminus. Mutated vs-1 elements were no longer bound by VSF-1 and also failed to activate the minimal -82 CaMV 35S promoter in vivo. Transient expression of VSF-1 in protoplasts stimulated vs-1 dependent activation of the -76/grp1.8 minimal promoter. Binding studies and use of a polyclonal antiserum against VSF-1 provided further evidence that vs-1 is a potential in vivo target site, as VSF-1 was a part of the observed complex formed between vs-1 and nuclear protein extract. vs-1 does not contain the 5'-ACGT-3' core sequence that is part of known plant bZIP protein binding sites or another palindromic sequence. Based on the unusual binding specificity and a characteristic amino acid sequence in the bZIP domain we propose that VSF-1 and the partially homologous PosF21, a bZIP protein from Arabidopsis, belong to a new family of plant bZIP proteins.

Amino Acid Sequence↗