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Hsiangfu Kung - One of the best experts on this subject based on the ideXlab platform.
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a nuclear matrix specific factor that binds a specific segment of the Negative Regulatory Element nre of hiv 1 ltr and inhibits nf κb activity
Nucleic Acids Research, 1996Co-Authors: Tammy Hoover, Judy A Mikovits, Dan Court, Yalun Liu, Hsiangfu KungAbstract:The Negative Regulatory Element (NRE) of human immunodeficiency virus type-1 (HIV-1) long terminal repeat (LTR) is a defined region that has been reported to downregulate LTR-directed HIV gene expression. However, information on the precise role of this region in regulating HIV gone transcription is lacking. We have investigated the possibility that these NRE sequences regulate HIV transcription by a mechanism mediated through a nuclear matrix-specific DNA-protein interaction. We find a nuclear matrix attachment region (MAR) present within the NRE of the HIV-1 LTR that recognizes a sequence-specific DNA-binding protein present in the nuclear matrix of HIV infected cells. Moreover, we also show that the purified DNA-binding nuclear matrix protein (NMP) specifically represses the DNA-binding activity of NF-kappaB. It is likely that the MAR and MAR-enriched specific DNA-binding NMP are brought into juxtaposition by the non-chromatin scaffolding of the nucleus, thus influencing NF-kappaB (and other nuclear proteins) DNA-binding activity through protein-protein and protein-DNA interactions. Our date suggest that one possible role of the NRE could be to act as a matrix attachment site in the nuclear matrix, thus, allowing interaction with a sequence-specific trans-acting factor. The Negative effect on NF-kappaB activity due to this MAR-NMP-specific interaction provides a mechanism by which the NRE downregulates HIV gene expression.
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characterization and purification of a novel transcriptional repressor from hela cell nuclear extracts recognizing the Negative Regulatory Element region of human immunodeficiency virus 1 long terminal repeat
Journal of Biological Chemistry, 1992Co-Authors: Michael West, Judy A Mikovits, Yalun Liu, Gerald L Princler, Francis W Ruscetti, Hsiangfu KungAbstract:Abstract Cellular transcription factors play critical roles in regulating human immunodeficiency virus (HIV) gene transcription, although the precise mechanism(s) defining their roles are not well established. Primarily it has been suggested that sequence-specific interaction of trans-activating proteins with cis-acting DNA Elements plays a crucial role in regulating the target genes. The Negative Regulatory Element (NRE) of HIV-1 long terminal repeat (LTR) is one such defined region that has been reported to down-regulate LTR-directed HIV gene expression. Information regarding the role of this region in the regulation of HIV expression is lacking. Here we describe an attempt to further characterize the role of NRE cis-Elements and define any sequence-specific interaction with cellular factors. Using gel mobility shift DNA-binding and Southwestern blot assays, we have mapped a distinct region of NRE (-290 to -260, a 30-base pair (bp) domain of NRE-A) sequences of HIV-1 LTR, which recognizes a specific DNA-binding protein from HeLa cell nuclear extracts. This factor is a 38-kDa polypeptide which can be affinity-purified to near homogeneity by this 30-bp specific oligonucleotide in affinity chromatography. The cellular factor from HeLa cell nuclear extract exhibits specific interaction only with the 30-bp NRE-A domain of HIV-1 LTR and acts as a strong transcriptional repressor/inhibitor molecule in the DNA-protein gel binding, as well as in vitro transcriptional studies with the nuclear extracts from cells with productive HIV-1 infection. To our knowledge, this is the first report of a factor recognizing a distinct segment within NRE that has been shown to exert an inhibitory effect on transcriptionally active DNA-protein "pre-initiation" complex formation, suggesting a possible role in HIV-1 gene regulation.
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cloning and characterization of a novel sequence specific dna binding protein recognizing the Negative Regulatory Element nre region of the hiv 1 long terminal repeat
Gene, 1991Co-Authors: Ida Calvert, Zequo Peng, Hsiangfu KungAbstract:Abstract The present studies describe the isolation of a murine cDNA clone that encodes a novel DNA-binding protein recognizing the Negative Regulatory Element ( NRE ) region of the HIV-1 long terminal repeat (LTR). This cDNA expresses a truncated protein with a functional DNA-binding domain, which is rich in glutamine/proline and serine/threonine, a characteristic of a majority of sequence-specific DNA-binding proteins and transcriptional factors. The cDNA hybridizes to a single-copy gene that is expressed as an approx. 4.2-kb mRNA in a variety of murine and human cell types, implying that this gene is expressed in an ubiquitous fashion. The NRE region has been reported to down-regulate LTR-directed gene expression [Rosen et al., Cell 41 (1985) 813–823]. This is the first sequence-specific DNA-binding protein reported to recognize the NRE region.
Ronald N Hines - One of the best experts on this subject based on the ideXlab platform.
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functional characterization of the human cyp1a1 Negative Regulatory Element modulation of ah receptor mediated transcriptional activity
Carcinogenesis, 1998Co-Authors: Marie P Piechocki, Ronald N HinesAbstract:The mechanisms that underly the regulation of human CYP1A1 have merited considerable attention because of their association both with toxic outcomes and the etiology of several cancers. Previous work conducted in this laboratory has identified a Negative Regulatory Element (NRE) in the 5' region of this gene that appeared to modulate CYP1A1 transcriptional activity. This NRE is present in two functional copies, a high affinity 21-bp palindrome centered at position -784, and an additional Element found within a GC-rich region between position -728 and -558. In this report, the Regulatory function of the NREs in the context of the CYP1A1 promoter was evaluated. This was accomplished by substituting mutated Elements for the corresponding wild-type Element in a vector that contained human CYP1A1 sequences positions -1140 to +59 directing the transcription of the chloramphenicol acetyltransferase reporter gene. Expression vectors containing specific mutations in each or both NREs were characterized. We show that eliminating the binding of the CYP1A1 repressor protein to one or both repressor motifs results in a significant 2- to 3-fold increase in the inducibility of CYP1A1 promoter activity. Although mutation of both sites appeared to result in an increase in inducibility over that observed with only one site mutated, the effect was not additive. Such aberrant transcriptional activity correlates with the highly inducible aryl hydrocarbon hydroxylase phenotype that is a reported marker for individuals predisposed to lung cancer. Mutation of the NRE, or more likely, the cognate repressor protein(s), may provide a genetic basis for this phenotype.
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in vitro binding and functional studies comparing the human cyp1a1 Negative Regulatory Element with the orthologous sequences from rodent genes
Carcinogenesis, 1995Co-Authors: Paul D Boucher, Ronald N HinesAbstract:In previous studies, we identified a 21 bp palindrome (-794 to -774) located within the Negative Regulatory Element of the human CYP1A1 gene consisting of an 8 bp inverted repeat and 5 bp spacer. This Element specifically binds protein(s) present in HepG2 nuclear extract preparations and is capable of down-regulating heterologous promoters and enhancers in transient expression assays. Conserved guanine/cytosine-rich regions which flank the palindrome also were implicated in this activity. In the present study, we examined similar regions from the rat (-881 to -746) and mouse (-822 to -683) CYP1A1 genes for their ability to bind nuclear protein and down-regulate heterologous promoters and enhancers. These rodent DNA fragments contain the conserved guanine/cytosine-rich sequences, as well as half-sites similar to those found in the human CYP1A1 palindrome. However, each half-site is separated by approximately 40 bp. DNase I footprint analyses revealed the presence of rat and mouse nuclear proteins which gave a similar protection pattern as that observed with nuclear proteins from the human cell line, HepG2. Electrophoretic mobility shift assays with the human Negative Regulatory Element demonstrated the formation of specific DNA-protein complexes with rat and mouse nuclear protein(s). Interestingly, two specific DNA-protein complexes were observed with rodent extracts as compared to the single specific complex seen with human extract. Specific binding was not observed with either the orthologous rat or mouse fragments using human or rodent extracts. In transient expression assays, the rat and mouse fragments were unable to down-regulate enhancer/promoter activity. This absence of Negative Regulatory activity occurred whether transfections were performed in human, rat or mouse hepatoma cell lines. The human Negative Regulatory Element, which was previously shown to down-regulate heterologous enhancers/promoters approximately 70% in human cells, did not exhibit this activity in rodent cell lines. UV cross-linking and southwestern blot analyses indicated a high degree of similarity between human and rodent NRE binding proteins, although some differences also were apparent. The possible implications of these findings with regards to species differences in the regulation of CYP1A1 expression are discussed.
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specific nuclear protein binding to a Negative Regulatory Element on the human cyp1a1 gene
Journal of Biological Chemistry, 1993Co-Authors: Paul D Boucher, R J Ruch, Ronald N HinesAbstract:Employing reporter gene/CYP1A1 chimeric plasmids, we previously identified a 275-base pair (bp) cis-Element (-833 to -558; NRE275) that down-regulated the CYP1A1 promoter. In the present study, this Negative Regulatory activity was further localized to two subfragments of 105 bp (-833 to -728; NRE105) and 170 bp (-728 to -558; NRE170), each of which exhibited activity with a heterologous promoter/enhancer. Co-transfection studies demonstrated a dependence on cellular trans-acting factors present at limiting concentrations. Electrophoretic mobility shift assays revealed the presence of protein(s) that specifically bound to NRE275, NRE105, and NRE170. Based upon competition studies, the protein(s) that bound to NRE105 appeared to recognize sites similar to those recognized by the NRE170-binding proteins. DNase I footprint analysis of NRE105 demonstrated nuclear protein binding to a 21-bp palindrome (-794 to -774). Protection was also observed along conserved guanine/cytosine-rich sequences that flank the palindrome, but in a strand-specific manner. Guanine residues involved in protein binding were identified by methylation interference experiments. Based on transient expression assays with each of the three NRE105 components, all three appear to be necessary for complete Negative Regulatory activity. However, it is clear the palindrome is the most important sequence with the guanine/cytosine-rich Elements playing an ancillary role.
David O. Peterson - One of the best experts on this subject based on the ideXlab platform.
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Identification of a protein that recognizes a distal Negative Regulatory Element within the mouse mammary tumor virus long terminal repeat.
Virology, 1999Co-Authors: Chang-joong Kang, David O. PetersonAbstract:Abstract The mouse mammary tumor virus (MMTV) long terminal repeat contains a distal Negative Regulatory Element (dNRE) that selectively represses activity of the proviral promoter in the absence of steroid hormone receptor-mediated activation. A protein, termed MMTV NRE-binding protein 1 (MNBP-1), that recognizes long terminal repeat sequences between −433 and −418 was identified by gel electrophoresis mobility shift assays and methylation interference footprinting in nuclear extracts of HeLa and Ltk − cells. Mutations within the defined binding site affect dNRE-mediated promoter repression in vivo. MNBP-1 has an apparent molecular mass of approximately 100 kDa as determined by gel filtration chromatography.
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multipartite structure of a Negative Regulatory Element associated with a steroid hormone inducible promoter
Journal of Biological Chemistry, 1991Co-Authors: Jae Woon Lee, P G Moffitt, K L Morley, David O. PetersonAbstract:The steroid hormone-inducible promoter from mouse mammary tumor virus is associated with a distal Negative Regulatory Element that represses its inherent basal activity. Deletion analysis localized the sequences required for repression to 64 base pairs of DNA between -427 and -364 with respect to the transcription initiation site. Transient transfection experiments with a series of linker scanning and small internal deletion mutations revealed two mutation-sensitive domains separated by a region of relative resistance to sequence alterations. DNase I footprinting and gel electrophoresis mobility shift experiments with crude nuclear extracts identified at least one protein-binding site within each of the two mutation-sensitive regions. An oligonucleotide corresponding to one of these sites is able to repress transcription, but only when linked to the promoter in multiple copies. This Negative Regulatory Element functions synergistically with a promoter proximal Negative Element to mediate efficient promoter repression, selectively affecting basal relative to steroid hormone-induced transcription and thus increasing the ratio of promoter activity observed in the presence and absence of hormone.
Yoshimasa Kyogoku - One of the best experts on this subject based on the ideXlab platform.
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identification of transcriptional suppressor proteins that bind to the Negative Regulatory Element of the human immunodeficiency virus type 1
Nucleic Acids Research, 1991Co-Authors: Kazuo Yamamoto, Shigehisa Mori, Takashi Okamoto, Kunitada Shimotohno, Yoshimasa KyogokuAbstract:Two different proteins which independently bound to neighboring sequences within the Negative Regulatory Element (NRE) of human immunodeficiency virus type 1 (HIV-1) were detected in the nuclear extract of a virus-infected human T cell line. One of the factors bound to a novel dyad symmetrical sequence. This sequence is well conserved in various HIV-1 isolates and partial homology was found with the promoter region of the human retinoblastoma gene. Similar DNA binding activity was detected in a variety of virus-uninfected human T cell lines and HeLa cells by means of a gel mobility shift assay. The other factor bound to a putative AP-1 recognition sequence predicted for the HIV-1 NRE. However, this factor did not bind to a typical AP-1 site. The insertion of multiple copies of the binding site for the former or latter factor into a heterologous promoter reduced the promoter activity to one-tenth or one-third, respectively. Thus, each factor may function as a novel Negative regulator of transcription.
Michael S Denison - One of the best experts on this subject based on the ideXlab platform.
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specificity of nuclear protein binding to a cyp1a1 Negative Regulatory Element
Biochemical and Biophysical Research Communications, 2002Co-Authors: Scott R Nagy, Michael S DenisonAbstract:Abstract Primary cultures of rat epidermal keratinocytes lose the ability to respond to chemicals with the induction of CYP1A1 gene expression after approximately 15 passages. This repression is mediated by a CT-rich direct repeat Negative Regulatory DNA (NeRD) Element present in the upstream Regulatory region of the CYP1A1 gene. Competitive gel retardation analysis using keratinocyte nuclear extracts and mutant NeRD oligonucleotides revealed the presence of two specific protein–NeRD complexes and revealed the specific nucleotides important for the formation of each complex. These studies demonstrate that these two factors bind to overlapping sites within the NeRD Element. Nucleotide specificity of complex A formation is similar to that of previously identified nuclear silencing factors, while that of complex B appears to represent a unique CT-rich binding factor. These results suggest that repression of CYP1A1 gene expression in high passage keratinocytes may involve the interplay between at least two specific NeRD binding factors.
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identification of a novel cis acting Negative Regulatory Element affecting expression of the cyp1a1 gene in rat epidermal cells
Journal of Biological Chemistry, 1996Co-Authors: Agnes A Walsh, Kathryn Tullis, Robert H Rice, Michael S DenisonAbstract:Polycyclic aromatic hydrocarbons such as 3-methylcholanthrene are toxic to rat epidermal cells in low passages (3 to 6), but cultures of high passage (≥15) are resistant. Since such compounds can be metabolically activated by cytochrome P4501A1, we have examined the regulation of this gene in low and high passage cells. Consistent with this difference, little or no 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible P4501A1 mRNA or enzyme activity was observed in high passage as compared to low passage cultures. Similarly, transfection of a luciferase reporter construct containing -1317 to +256 base pairs of the 5′-flanking region of the murine CYP1A1 gene was TCDD-inducible in low but not high passage cells. Ligand binding and transfection experiments demonstrated the presence of functional Ah receptor complexes in both high and low passage cells. Deletion analysis identified a 26-base pair Negative Regulatory DNA (NeRD) Element contained within the upstream Regulatory region of the CYP1A1 gene responsible for this effect. Nuclear extracts from both low and high passage cells contain a protein which specifically binds to NeRD-containing DNA. Thus, the loss of polycyclic aromatic hydrocarbon sensitivity in high passage rat epidermal cells appears to be due to decreased expression of CYP1A1, and this effect may be mediated by an altered NeRD binding factor(s) present in these cells.
