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David Saffen - One of the best experts on this subject based on the ideXlab platform.
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expression of the rat m4 muscarinic acetylcholine receptor gene is regulated by the neuron restrictive Silencer Element repressor Element 1
Journal of Biological Chemistry, 1997Co-Authors: Michihiro Mieda, Tatsuya Haga, David SaffenAbstract:Neuronal cell-specific expression of the rat m4 muscarinic acetylcholine receptor (mAChR) is regulated by a Silencer Element. A likely mediator of this silencing is the neuron-restrictive Silencer Element/repressor Element 1 (NRSE/RE1), which is present 837 base pairs (bp) upstream from the transcription initiation site of the m4 mAChR gene (Wood, I. C., Roopra, A., Harrington, C., and Buckley, N. J. (1995) J. Biol. Chem. 270, 30933-30940; Mieda, M., Haga, T., and Saffen, D. W. (1996) J. Biol. Chem. 271, 5177-5182). In the present study, we examined whether this putative NRSE/RE1 functions as a Silencer. Transient expression assays using m4 mAChR promoter/luciferase expression vectors showed that the m4 NRSE/RE1 is necessary and sufficient to repress m4 promoter activity in non-neuronal L6 cells. m4 promoter activity was only partially repressed, however, in neuronal NG108-15 cells exogenously expressing the neuronal-restrictive Silencer factor/RE1-silencing transcription factor (NRSF/REST). By contrast, the promoter activity of the type II sodium channel (NaII) gene was nearly completely repressed in NRSF/REST-expressing NG108-15 cells. Experiments with expression vectors containing chimeric promoters revealed that the NRSE/RE1 Elements derived from both the m4 and NaII genes are independently sufficient to silence NaII gene promoter activity, but only partially repress m4 mAChR gene promoter activity in NRSF/REST-expressing NG108-15 cells. Thus, the repression activity of NRSF/REST depends upon the species of promoter to which it is linked. Gel-shift assays showed that the NRSF/REST is the only protein that binds to a 92-bp segment from the m4 mAChR promoter containing NRSE/RE1. This and the fact that m4 promoter activity was completely repressed in L6 cells suggest that the proteins that bind to the m4 constitutive promoter may be different from those in NG108-15 cells. Deletion analysis of the m4 constitutive promoter revealed that a 90-bp segment immediately upstream from the transcription initiation site contains significant promoter activity. Gel-shift assays revealed that several proteins in nuclear extracts prepared from L6 and NG108-15 cells bind to this 90-bp segment and that some of these proteins are L6 or NG108-15 cell-specific. These data support the idea that the repression activity of NRSF/REST depends upon the species of promoter to which it is linked and upon the proteins that bind to those promoters.
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promoter region of the rat m4 muscarinic acetylcholine receptor gene contains a cell type specific Silencer Element
Journal of Biological Chemistry, 1996Co-Authors: Michihiro Mieda, Tatsuya Haga, David SaffenAbstract:We describe here the characterization of the rat m4 muscarinic acetylcholine receptor gene and the identification of its regulatory region. Two 5′-noncoding exons are located approximately 5 kilobases upstream from the coding exon, and at least two alternatively spliced variants of m4 mRNA are expressed in the neuronal cell line PC12D. There are two transcription initiation sites. The promoter region is GC-rich, contains no TATA-box, but has two potential CAAT boxes and several putative binding sites for transcription factors Sp1 and AP-2. We assessed the m4 promoter activity functionally in transient expression assays using luciferase as a reporter. The proximal 435-base pair (bp) sequence of the 5′-flanking region produced luciferase activity in both m4-expressing neuronal cell lines (PC12D and NG108-15) and non-neuronal cell lines (L6 and 3Y1B). A longer fragment containing an additional 638-bp sequence produced luciferase activity only in m4-expressing neuronal cell lines. These data suggest that the proximal 435-bp sequence contains a constitutive promoter and that a 638-bp sequence farther upstream contains a cell type-specific Silencer Element. A consensus sequence for the neural-restrictive Silencer Element is found within this 638-bp segment.
Michihiro Mieda - One of the best experts on this subject based on the ideXlab platform.
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expression of the rat m4 muscarinic acetylcholine receptor gene is regulated by the neuron restrictive Silencer Element repressor Element 1
Journal of Biological Chemistry, 1997Co-Authors: Michihiro Mieda, Tatsuya Haga, David SaffenAbstract:Neuronal cell-specific expression of the rat m4 muscarinic acetylcholine receptor (mAChR) is regulated by a Silencer Element. A likely mediator of this silencing is the neuron-restrictive Silencer Element/repressor Element 1 (NRSE/RE1), which is present 837 base pairs (bp) upstream from the transcription initiation site of the m4 mAChR gene (Wood, I. C., Roopra, A., Harrington, C., and Buckley, N. J. (1995) J. Biol. Chem. 270, 30933-30940; Mieda, M., Haga, T., and Saffen, D. W. (1996) J. Biol. Chem. 271, 5177-5182). In the present study, we examined whether this putative NRSE/RE1 functions as a Silencer. Transient expression assays using m4 mAChR promoter/luciferase expression vectors showed that the m4 NRSE/RE1 is necessary and sufficient to repress m4 promoter activity in non-neuronal L6 cells. m4 promoter activity was only partially repressed, however, in neuronal NG108-15 cells exogenously expressing the neuronal-restrictive Silencer factor/RE1-silencing transcription factor (NRSF/REST). By contrast, the promoter activity of the type II sodium channel (NaII) gene was nearly completely repressed in NRSF/REST-expressing NG108-15 cells. Experiments with expression vectors containing chimeric promoters revealed that the NRSE/RE1 Elements derived from both the m4 and NaII genes are independently sufficient to silence NaII gene promoter activity, but only partially repress m4 mAChR gene promoter activity in NRSF/REST-expressing NG108-15 cells. Thus, the repression activity of NRSF/REST depends upon the species of promoter to which it is linked. Gel-shift assays showed that the NRSF/REST is the only protein that binds to a 92-bp segment from the m4 mAChR promoter containing NRSE/RE1. This and the fact that m4 promoter activity was completely repressed in L6 cells suggest that the proteins that bind to the m4 constitutive promoter may be different from those in NG108-15 cells. Deletion analysis of the m4 constitutive promoter revealed that a 90-bp segment immediately upstream from the transcription initiation site contains significant promoter activity. Gel-shift assays revealed that several proteins in nuclear extracts prepared from L6 and NG108-15 cells bind to this 90-bp segment and that some of these proteins are L6 or NG108-15 cell-specific. These data support the idea that the repression activity of NRSF/REST depends upon the species of promoter to which it is linked and upon the proteins that bind to those promoters.
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promoter region of the rat m4 muscarinic acetylcholine receptor gene contains a cell type specific Silencer Element
Journal of Biological Chemistry, 1996Co-Authors: Michihiro Mieda, Tatsuya Haga, David SaffenAbstract:We describe here the characterization of the rat m4 muscarinic acetylcholine receptor gene and the identification of its regulatory region. Two 5′-noncoding exons are located approximately 5 kilobases upstream from the coding exon, and at least two alternatively spliced variants of m4 mRNA are expressed in the neuronal cell line PC12D. There are two transcription initiation sites. The promoter region is GC-rich, contains no TATA-box, but has two potential CAAT boxes and several putative binding sites for transcription factors Sp1 and AP-2. We assessed the m4 promoter activity functionally in transient expression assays using luciferase as a reporter. The proximal 435-base pair (bp) sequence of the 5′-flanking region produced luciferase activity in both m4-expressing neuronal cell lines (PC12D and NG108-15) and non-neuronal cell lines (L6 and 3Y1B). A longer fragment containing an additional 638-bp sequence produced luciferase activity only in m4-expressing neuronal cell lines. These data suggest that the proximal 435-bp sequence contains a constitutive promoter and that a 638-bp sequence farther upstream contains a cell type-specific Silencer Element. A consensus sequence for the neural-restrictive Silencer Element is found within this 638-bp segment.
David J Anderson - One of the best experts on this subject based on the ideXlab platform.
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cell type specific regulation of choline acetyltransferase gene expression role of the neuron restrictive Silencer Element and cholinergic specific enhancer sequences
Journal of Biological Chemistry, 1996Co-Authors: Peter Lonnerberg, Christopher J Schoenherr, David J Anderson, Carlos F IbanezAbstract:This study demonstrates the presence of positive and negative regulatory Elements within a 2336-base pair-long region of the rat choline acetyltransferase (ChAT) gene promoter that cooperate to direct cell type-specific expression in cholinergic cells. A 21-base pair-long neuron-restrictive Silencer Element (NRSE) was identified in the proximal part of this region. This Element was recognized by the neuron-restrictive Silencer factor (NRSF), previously shown to regulate expression of other neuron-specific genes. The ChAT NRSE was inactive in both cholinergic and non-cholinergic neuronal cells, but repressed expression from a heterologous promoter in non-neuronal cells. Specific deletion of this Element allowed ChAT gene promoter activity in non-neuronal cells, and overexpression of NRSF repressed ChAT gene promoter activity in cholinergic cells. The distal part of the ChAT gene promoter showed cholinergic-specific enhancing activity, which stimulated promoter activity in cholinergic cells, but was inactive in non-cholinergic neuronal and non-neuronal cells. This enhancer region suppressed the activity of the ChAT NRSE in cholinergic cells, even after NRSF overexpression. Thus, at least two kinds of regulatory Elements cooperate to direct ChAT gene expression to cholinergic neurons, namely a neuron-restrictive Silencer Element and a cholinergic-specific enhancer.
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identification of potential target genes for the neuron restrictive Silencer factor
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Christopher J Schoenherr, Alice J Paquette, David J AndersonAbstract:The neuron-restrictive Silencer factor (NRSF) represses transcription of several neuronal genes in nonneuronal cells by binding to a 21-bp Element called the neuron-restrictive Silencer Element (NRSE). We have performed data base searches with a composite NRSE to identify additional candidate NRSF target genes. Twenty-two more genes, 17 of which are expressed mainly in neurons, were found to contain NRSE-like sequences. Many of these putative NRSEs bound NRSF in vitro and repressed transcription in vivo. Most of the neuronal genes identified contribute to the basic structural or functional properties of neurons. However, two neuronal transcription factor genes contain NRSEs, suggesting that NRSF may repress neuronal differentiation both directly and indirectly. Functional NRSEs were also found in several nonneuronal genes, implying that NRSF may play a broader role than originally anticipated.
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the neuron restrictive Silencer factor nrsf a coordinate repressor of multiple neuron specific genes
Science, 1995Co-Authors: Christopher J Schoenherr, David J AndersonAbstract:The neuron-restrictive Silencer factor (NRSF) binds a DNA sequence Element, called the neuron-restrictive Silencer Element (NRSE), that represses neuronal gene transcription in nonneuronal cells. Consensus NRSEs have been identified in 18 neuron-specific genes. Complementary DNA clones encoding a functional fragment of NRSF were isolated and found to encode a novel protein containing eight noncanonical zinc fingers. Expression of NRSF mRNA was detected in most nonneuronal tissues at several developmental stages. In the nervous system, NRSF mRNA was detected in undifferentiated neuronal progenitors, but not in differentiated neurons. NRSF represents the first example of a vertebrate Silencer protein that potentially regulates a large battery of cell type-specific genes, and therefore may function as a master negative regulator of neurogenesis.
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a common Silencer Element in the scg10 and type ii na channel genes binds a factor present in nonneuronal cells but not in neuronal cells
Neuron, 1992Co-Authors: Nozomu Mori, Christopher J Schoenherr, David J Vandenbergh, David J AndersonAbstract:We have localized a cell type-specific Silencer Element in the SCG10 gene by deletion analysis. This neural-restrictive Silencer Element (NRSE) selectively represses SCG10 expression in nonneuronal cells and tissues. The NRSE contains a 21 by region with striking homology to a sequence present in a Silencer domain of the rat type II sodium channel (Nall), another neuron-specific gene. We have identified a sequence-specific protein(s) that binds the SCG10 NRSE, as well as the homologous Element in the Nall gene. A point mutation in the NRSE that abolishes binding of this neural-restrictive Silencerbinding factor (NRSBF) in vitro also eliminates silencing activity in vivo. NRSBF is present in nuclear extracts from nonneuronal cells but not in extracts from neuronal cells, suggesting that the neuron-specific expression of SCG10 reflects, at least in part, the absence or inactivity of this protein. These data identify the NRSE as a potentially general DNA Element for the control of neuron-specific gene expression in vertebrates.
Laurence H Hurley - One of the best experts on this subject based on the ideXlab platform.
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molecular modeling and biophysical analysis of the c myc nhe iii1 Silencer Element
Journal of Molecular Modeling, 2008Co-Authors: Derek J Cashman, Laurence H Hurley, Robert Buscaglia, Matthew W Freyer, Jamie M Dettler, Edwin A LewisAbstract:G-Quadruplex and i-Motif-forming sequences in the promoter regions of several oncogenes show promise as targets for the regulation of oncogenes. In this study, molecular models were created for the c-MYC NHE-III1 (nuclease hypersensitivity Element III1) from two 39-base complementary sequences. The NHE modeled here consists of single folded conformers of the polypurine intramolecular G-Quadruplex and the polypyrimidine intramolecular i-Motif structures, flanked by short duplex DNA sequences. The G-Quadruplex was based on published NMR structural data for the c-MYC 1:2:1 loop isomer. The i-Motif structure is theoretical (with five cytosine–cytosine pairs), where the central intercalated cytosine core interactions are based on NMR structural data obtained for a tetramolecular [d(A2C4)4] model i-Motif. The loop structures are in silico predictions of the c-MYC i-motif loops. The porphyrin meso-tetra(N-methyl-4-pyridyl)porphine (TMPyP4), as well as the ortho and meta analogs TMPyP2 and TMPyP3, were docked to six different locations in the complete c-MYC NHE. Comparisons are made for drug binding to the NHE and the isolated G-Quadruplex and i-Motif structures. NHE models both with and without bound cationic porphyrin were simulated for 100 ps using molecular dynamics techniques, and the non-bonded interaction energies between the DNA and porphyrins calculated for all of the docking interactions.
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drug targeting of the c myc promoter to repress gene expression via a g quadruplex Silencer Element
Seminars in Oncology, 2006Co-Authors: Laurence H Hurley, Daniel D Von Hoff, Adam Siddiquijain, Danzhou YangAbstract:In this review, we describe the evidence for a parallel-stranded G-quadruplex in the purine-rich strand of the nuclease hypersensitivity Element III 1 (NHE III 1 ) of the promoter of c-MYC upstream of the P1 and P2 promoters. This biologically relevant G-quadruplex is a mixture of four loop isomers. The folding pattern of a nuclear magnetic resonance (NMR)-derived structure for the predominant loop isomer of this G-quadruplex has been obtained. This G-quadruplex has been demonstrated to be a Silencer Element, and the cationic porphyrin TMPyP4 has been shown to stabilize this G-quadruplex. Furthermore, TMPyP4 has been shown to repress c-MYC expression, and this effect is mediated through the Silencer Element. Last, the in vivo activity of TMPyP4 in xenograph models is presented.
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structure of the biologically relevant g quadruplex in the c myc promoter
Nucleosides Nucleotides & Nucleic Acids, 2006Co-Authors: Danzhou Yang, Laurence H HurleyAbstract:The nuclease hypersensitivity Element III1 (NHE III1) in the c-MYC promoter controls up to 80-90% of the transcriptional activity of this gene. We have demonstrated that the guanine-rich strand of the NHE III1 forms a G-quadruplex consisting of a mixture of four biologically relevant loop isomers that function as a Silencer Element. NMR studies have shown that these G-quadruplexes are propeller-type parallel structures consisting of three stacked G-tetrads and three double-chain reversal loops. An NMR-derived solution structure for this quadruplex provides insight into the unusual stability of the structure. This structure is a target for small molecule inhibitors of c-MYC gene expression.
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retraction for grand et al mutations in the g quadruplex Silencer Element and their relationship to c myc overexpression nm23 repression and therapeutic rescue pnas 2004 101 6140 6145
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Cory L Grand, Tiffanie J Powell, Raymond B Nagle, David J Bearss, Denise Tye, Mary Gleasonguzman, Laurence H HurleyAbstract:MEDICAL SCIENCES. For the article “Mutations in the G-quadruplex Silencer Element and their relationship to c-MYC overexpression, NM23 repression, and therapeutic rescue,” by Cory L. Grand, Tiffanie J. Powell, Raymond B. Nagle, David J. Bearss, Denise Tye, Mary Gleason-Guzman, and Laurence H. Hurley, …
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mutations in the g quadruplex Silencer Element and their relationship to c myc overexpression nm23 repression and therapeutic rescue
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Cory L Grand, Tiffanie J Powell, Raymond B Nagle, David J Bearss, Denise Tye, Mary Gleasonguzman, Laurence H HurleyAbstract:We have demonstrated that a parallel G-quadruplex structure in the c-MYC promoter functions as a transcriptional repressor Element. Furthermore, a specific G-to-A mutation in this Element results in destabilization of the G-quadruplex repressor Element and an increase in basal transcriptional activity. To validate this model in an in vivo context, we have examined the sequence of this region in human colorectal tumors and the surrounding normal tissue. We have found that ≈30% of tumors contain one of two specific G-to-A mutations, not present in the surrounding normal tissue, that destabilize the parallel G-quadruplex, which would be expected to give rise to abnormally high expression of c-MYC in these cells. In contrast, G-quadruplex-disruptive mutations were absent in 20 colon adenomas, suggesting that these mutations occur late in tumorigenesis. We have also demonstrated that these same mutations are found in established colorectal cell lines. NM23-H2 levels are lower in cancer tissues and cell lines that harbor these mutations. In cells with repressed levels of NM23-H2, the mutated and destabilized G-quadruplex Silencer Element can be reinstated by the addition of G-quadruplex-stabilizing compounds, providing an opportunity for therapeutic intervention for patients carrying these mutations.
Peter D Nagy - One of the best experts on this subject based on the ideXlab platform.
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role of an internal and two 3 terminal rna Elements in assembly of tombusvirus replicase
Journal of Virology, 2005Co-Authors: Zivile Panaviene, Tadas Panavas, Peter D NagyAbstract:, which also coexpressed p33 andp92 replication proteins. In vitro replicase assays performed with purified CNV replicase preparations fromyeast revealed critical roles for three RNA Elements in CNV replicase assembly: the internal p33 recognitionElement (p33RE), the replication Silencer Element (RSE), and the 3 -terminal minus-strand initiation pro-moter (gPR). Deletion or mutagenesis of these Elements reduced the activity of the CNV replicase to a minimallevel. In addition to the primary sequences of gPR, RSE, and p33RE, formation of two alternative structuresamong these Elements may also play a role in replicase assembly. Altogether, the role of multiple RNA Elementsin tombusvirus replicase assembly could be an important factor to ensure fidelity of template selection duringreplication.
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role of an internal and two 3 terminal rna Elements in assembly of tombusvirus replicase
Journal of Virology, 2005Co-Authors: Zivile Panaviene, Tadas Panavas, Peter D NagyAbstract:, which also coexpressed p33 andp92 replication proteins. In vitro replicase assays performed with purified CNV replicase preparations fromyeast revealed critical roles for three RNA Elements in CNV replicase assembly: the internal p33 recognitionElement (p33RE), the replication Silencer Element (RSE), and the 3 -terminal minus-strand initiation pro-moter (gPR). Deletion or mutagenesis of these Elements reduced the activity of the CNV replicase to a minimallevel. In addition to the primary sequences of gPR, RSE, and p33RE, formation of two alternative structuresamong these Elements may also play a role in replicase assembly. Altogether, the role of multiple RNA Elementsin tombusvirus replicase assembly could be an important factor to ensure fidelity of template selection duringreplication.
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a replication Silencer Element in a plus strand rna virus
The EMBO Journal, 2003Co-Authors: Judit Pogany, Marc R Fabian, Andrew K White, Peter D NagyAbstract:Replication represents a key step in the infectious cycles of RNA viruses. Here we describe a regulatory RNA Element, termed replication Silencer, that can down-regulate complementary RNA synthesis of a positive-strand RNA virus via an RNA–RNA interaction. This interaction occurs between the 5-nucleotide-long, internally positioned replication Silencer and the extreme 3′-terminus of the viral RNA comprising part of the minimal minus-strand initiation promoter. Analysis of RNA synthesis in vitro, using model defective interfering (DI) RNA templates of tomato bushy stunt virus and a partially purified, RNA-dependent RNA polymerase preparation from tombusvirus-infected plants, revealed that this interaction inhibits minus-strand synthesis 7-fold. This functional interaction was supported further by: (i) RNA structure probing; (ii) phylogenetic analysis; (iii) inhibition of activity by short complementary DNAs; and (iv) compensatory mutational analysis. The Silencer was found to be essential for accumulation of DI RNAs in protoplasts, indicating that it serves an important regulatory role(s) in vivo. Because similar Silencer–promoter interactions are also predicted in other virus genera, this type of RNA-based regulatory mechanism may represent a widely utilized strategy for modulating replication.