DrugBank:BIOD00001 - FACTA Search

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Query: DrugBank:BIOD00001 (DNase I)
8,324 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Evidence indicates DNA methylation as a part of the regulatory machinery controlling mammalian gene expression. The human melanoma cell line HA-A expresses low levels of transforming growth factor alpha (TGF-alpha). TGF-alpha mRNA accumulated, however, in response to DNA demethylation induced by a nucleoside analog, 5-azacytidine (5-azaC). The importance of DNA methylation in the TGF-alpha promoter region was examined by a transient transfection assay with luciferase reporter plasmids containing a portion of the TGF-alpha promoter. 5-azaC treatment of HA-A cells before the transfection caused a significant increase in the luciferase activity. Since input plasmids were confirmed to remain unmethylated, DNA demethylation of the TGF-alpha promoter itself does not account for the observed increase in TGF-alpha mRNA. Using an electrophoretic mobility shift assay, enhanced formation of protein-TGF-alpha promoter complex was detected in response to 5-azaC treatment. This 5-azaC-induced complex was shown to contain the transcription factor Sp1 by the following criteria: the protein-DNA complex formed on the TGF-alpha promoter contained immunoreactive Sp1; the mobility of the complex in an electrophoretic mobility shift assay was similar to that formed by recombinant Sp1; and DNase I footprinting analysis demonstrated that the 5-azaC-induced complex produced a footprint on the TGF-alpha promoter identical to that of authentic Sp1. These observations suggest that 5-azaC induces TGF-alpha expression by augmenting the Sp1 activity. However, neither the Sp1 mRNA nor its protein was induced by 5-azaC. These results suggest that in HA-A cells, TGF-alpha expression is down-modulated by DNA methylation. In addition, this process may involve the specific regulation of Sp1 activity without altering the amount of the transcription factor.
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PMID:5-Azacytidine treatment of HA-A melanoma cells induces Sp1 activity and concomitant transforming growth factor alpha expression. 138 Jun 48

The insulin-like growth factor I receptor (IGF-I-R) gene is expressed in most body tissues. The levels of IGF-I-R mRNA, however, are regulated by a number of physiological conditions (development, differentiation, and hormonal milieu) as well as in certain pathological states (diabetes and tumors). To understand the molecular mechanisms which control the transcription of the IGF-I-R gene, we have cloned the promoter of the rat receptor gene and have characterized its activity by transient expression assays. Different fragments of the 5'-flanking region (subcloned upstream of a luciferase reporter gene) were transfected into buffalo rat liver 3A cells (a cell line with a low number of IGF-I binding sites) and Chinese hamster ovary cells (a cell line with a higher number of cell-surface receptors). In both cell lines, most of the promoter activity was located in the proximal 416 base pairs of 5'-flanking region. However, further dissection of this proximal fragment revealed a cell type-specific pattern of promoter activity. Thus, in buffalo rat liver 3A cells, subfragments of this region each contributed to total activity, suggesting that contiguous cis-elements can act together to activate transcription. In Chinese hamster ovary cells, on the other hand, subfragments of the proximal promoter region partially substituted for the proximal 416 base pairs of 5'-flanking region. Coexpression studies using an IGF-I-R promoter reporter construct together with an Sp1 expression vector (under the control of an ADH promoter) were performed in SL2 cells, a Drosophila cell line which lacks endogenous Sp1. The results obtained showed that Sp1 can trans-activate the IGF-I-R promoter in vivo. Transient transfection assays were complemented with gel-retardation assays and DNase I footprinting experiments, which showed that transcription factor Sp1 is potentially an important regulator of IGF-I-R gene expression.
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PMID:Structural and functional analysis of the insulin-like growth factor I receptor gene promoter. 144 10

The gene encoding mZP3, the mouse sperm receptor, is expressed exclusively in growing oocytes during oogenesis. To investigate the molecular basis of oocyte-specific mZP3 gene expression, we generated several lines of mice harboring a transgene that contains 470 bp of mZP3 gene 5'-flanking sequence (nucleotides -470 to +10) fused to the firefly luciferase gene coding region. Three of four expressing transgenic lines exhibited luciferase activity only in growing oocytes, suggesting that the 470-bp fragment is sufficient to direct Iocyte-specific expression of the luciferase gene. Results of DNase I footprinting and gel mobility shift assays suggested the presence of an ovary-specific protein that binds to a small region (nucleotides-99 to -86) within the 470-bp fragment of the mZP3 promoter, with 5'-G(G/A)T(G/A)A-3' representing the minimal sequence required for binding. Southwestern (DNA-protein) gel blots revealed the presence of an oocyte-specific, approximately 60,000-Mr protein, called OSP-1, that binds to the minimal sequence. Changes in levels of OSP-1 during oogenesis and early cleavage are consistent with the pattern of mZP3 gene expression during these developmental stages in mice. Therefore, OSP-1 may be a mammalian oocyte-specific transcription factor involved in regulating oocyte-specific mZP3 gene expression.
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PMID:A mouse oocyte-specific protein that binds to a region of mZP3 promoter responsible for oocyte-specific mZP3 gene expression. 172 94

Lipoprotein lipase (LPL), a key enzyme in normal lipoprotein metabolism, has a complex pattern of regulation and tissue-specific expression. Several potential binding sites for transcription factors, including the recognition sequences for CCAAT/enhancer-binding protein and octamer-binding proteins (Oct) have been described in the 5'-flanking region of the human LPL gene. To identify elements which regulate the expression of LPL in adipocytes, plasmids containing deletion mutants of the 5'-LPL promoter region and the luciferase reporter gene were transfected in 3T3-L1 adipocytes. Deletions at -724, -565, -461, -368, -232, -167, -92, -35, and -17 relative to the transcriptional start site modified transcription from 100 to 162, 194, 185, 128, 63, 53, 29, and 0%, respectively, indicating the presence of negative (-724 to -565) and positive (-368 to -35) cis-acting regulatory elements. Transfection of HepG2 cells, which do not synthesize LPL, with the same constructs resulted in a similar pattern of expression for the majority of the deletions. However, deletions between -724 and -368 base pairs resulted in a 75-100% increase in transcription in 3T3 adipocytes but not in HepG2 cells, indicating the presence of tissue-specific regulatory element(s) in this region. An important regulatory element affecting LPL transcription in adipocytes was identified by gel mobility shift assays and DNase I footprint analysis. Using these techniques, a nuclear protein(s) in 3T3-L1 adipocytes was shown to bind specifically to a fragment which included the proximal octamer recognition site (from -46 to -39) present in the LPL promoter. The DNA-protein complex comigrates with an electrophoretic band containing the Oct-1-DNA complex in BJA-B nuclear extracts and the DNA-protein complex was selectively competed only by DNA fragments containing the octamer sequence. Preincubation of 3T3-L1 nuclear extracts with an antibody directed against the POU domain of Oct-1 inhibited the formation of the DNA-protein complex. Deletion of the proximal octanucleotide motif from the plasmid containing the -461 fragment of the LPL promoter, resulted in a 79 and 76% decrease in the level of expression in transfected 3T3-L1 adipocytes and HepG2 hepatocytes, respectively. These combined results have established that the expression of LPL in adipocytes is modulated by multiple positive and negative regulatory elements within the 5'-flanking region of the LPL gene. A proximal octamer binding sequence which specifically interacts with a nuclear protein(s) that exhibits the characteristics of Oct-1 has been identified.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Transcriptional regulation of the human lipoprotein lipase gene in 3T3-L1 adipocytes. 191 10

The capacity of cells of the human bronchial epithelium to express the gene for interleukin-8 (IL-8) was evaluated in bronchial epithelium derived cell lines, HS-24 and BET-1A, using tumor necrosis factor-alpha (TNF) as a model inflammatory stimulus. As in other epithelium, TNF markedly increased the level of the 1.8-kilobase IL-8 mRNA transcripts in both bronchial epithelial cell lines. In HS-24 cells, nuclear run-on analyses showed the IL-8 gene transcription rate was dramatically increased, more than 30-fold, after TNF stimulation. The half-life of IL-8 mRNA transcripts in these cells was approximately 40 min and did not change after TNF stimulation, suggesting that TNF up-regulated IL-8 gene expression mainly at the transcriptional level. DNase I hypersensitivity site mapping of chromatin DNA in resting HS-24 cells demonstrated two hypersensitivity sites within 400 base pairs (bp) 5' to exon I and one site within exon I. However, after TNF stimulation, the exon I hypersensitivity site disappeared and a new site approximately 120 bp 5' to exon I emerged. Consistent with these observations, transfection studies with HS-24 cells using fusion genes composed of the 5'-flanking sequences of the IL-8 gene and a luciferase reporter gene demonstrated potent promoter activity in a 174-bp segment (-130 to +44 relative to the transcription start site), which also exhibited a response to TNF, while a segment from -112 to +44 showed very low promoter activity and no response to TNF. Thus, human bronchial epithelial cells can express the IL-8 gene, with expression in response to the inflammatory mediator TNF regulated mainly at the transcriptional level, and with elements within the 5'-flanking region of the gene that are directly or indirectly modulated by the TNF signal.
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PMID:Interleukin-8 gene expression in human bronchial epithelial cells. 191 68

This study is an attempt to determine whether estrogen could directly regulate human gonadotropin-releasing hormone (GnRH) gene expression. Human GnRH expression vectors were constructed by fusing various 5' flanking regions of the human GnRH gene upstream of the luciferase reporter gene (LUC) or the thymidine kinase promoter linked to the chloramphenicol acetyltransferase reporter gene (CAT). These constructs were transiently transfected into a human choriocarcinoma cell line (JEG-3) and LUC or CAT activity was measured after either no treatment or treatment with various concentrations of estradiol. A stimulatory estrogen response element (ERE) was localized to a 32-bp region between -547 and -516 bp. To determine whether estrogen receptor bound to this region of the gene, we performed DNase I footprinting using purified calf uterine estrogen receptor. DNase I footprinting demonstrates a strong footprint between -567 and -514 bp of the human GnRH gene. In addition, an avidin-biotin complex DNA-binding assay demonstrated that a biotinylated DNA fragment containing -541 to -517 bp of the human GnRH gene bound 35S-labeled estrogen receptor as well as a biotinylated DNA fragment containing the xenopus vitellogenin ERE. On the other hand, the negative control biotinylated DNA fragment derived from adenovirus 5 bound insignificant amounts of 35S-labeled estrogen receptor. Both the GnRH ERE and vitellogenin ERE bound 35S-labeled estrogen receptor with high affinity (approximately 1 nM). These data indicate that the human GnRH gene contains an ERE sufficient to mediate a stimulatory response to estrogen in heterologous cells. Based upon these data we hypothesize that the human GnRH gene might also be directly regulated by estrogen in the hypothalamus, and that this regulation may explain the GnRH hypersecretion observed at the time of the preovulatory luteinizing hormone (LH) surge.
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PMID:Evidence for direct estrogen regulation of the human gonadotropin-releasing hormone gene. 193 51

The rat angiotensinogen gene is induced in the course of the hepatic acute-phase response. We demonstrate that monocyte conditioned medium can stimulate transcription of a stably introduced reporter construct driven by 615 base pairs of the angiotensinogen 5'-flanking sequence, as well as the endogenous gene, in Reuber H35 cells. Point mutations of a cis-acting element, located 545 base pairs from the transcription start site and sharing sequence identity with known nuclear factor kappa B (NF kappa B)-binding sites, led to loss of cytokine inducibility. When cloned upstream of a minimal promoter, this cis-acting element imparted transcriptional inducibility by monocyte conditioned medium, interleukin-1, and tumor necrosis factor on a luciferase reporter gene in HepG2 cells. Two distinct proteins bound this element in vitro: a heat-stable, constitutively present, hepatic nuclear protein that gave rise to a DNase I-protected footprint covering the functionally defined element; and a binding protein of different mobility, induced by monocyte conditioned medium, which also recognized the NF kappa B-binding site of the murine kappa light-chain enhancer. UV cross-linking showed this inducible protein to have an apparent molecular mass of 50 kilodaltons, similar to that described for NF kappa B and distinct from the constitutively present protein that was shown by Southwestern (DNA-protein) blot to have a molecular mass of 32 kilodaltons. Methylation interference analysis showed that the induced species made contact points with guanine residues in the NF kappa B consensus sequence typical of NF kappa B. Induction of this binding activity did not require new protein synthesis, and 12-O-tetradecanoylphorbol-13-acetate could mimic the induction by cytokines. We thus provide direct evidence for involvement of NF kappa B or a similar factor in the hepatic acute-phase response and discuss the potential role of the presence of a constitutive nuclear factor binding the same cis element.
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PMID:An inducible 50-kilodalton NF kappa B-like protein and a constitutive protein both bind the acute-phase response element of the angiotensinogen gene. 210 65

The murine thyrotropic MGH101A tumor is characterized by absent thyrotropin (TSH) beta gene expression and altered thyroid hormone (T3) regulation of the alpha-subunit. Comparison of the promoter structures of both alpha and TSH beta subunit genes from MGH101A with the promoter in expressing TtT-97 thyrotropes revealed no detectable differences. Transfection of the TSH beta promoter from MGH101A linked to luciferase showed minimal expression in primary or cloned MGH101A cells, or L-cells. However, a 6- to 10-fold increase in expression was exhibited in transfected thyrotropes. For the alpha gene, promoter activity was highest in thyrotropes and in cloned MGH101A cells, 5-fold lower in MGH101A tumors, and 10-fold lower in L-cells. Both promoters were not substantially affected by T3 treatment in MGH101A cells. In thyrotropes, promoter activity was inhibited 62.5% and 57.7% by 10 nM T3 treatment for the TSH beta and alpha genes, respectively. DNase I protection showed that factors from TtT-97 but not from MGH101A cells interacted with regions in the TSH beta promoter, while nuclear extracts from each tumor demonstrated at least one protein-DNA interaction with the alpha-subunit promoter. These studies suggest that the molecular defects in the MGH101A tumor are related to the absence of trans-acting factors and are not a result of altered primary gene structure.
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PMID:TSH subunit gene promoters from a murine alpha-subunit producing tumor function normally. 237 87

Pituitary thyrotroph cells specialize in the synthesis of TSH, and thus represent a model to study cell-specific gene expression. We have used the murine TSH beta (mTSH beta) gene promoter and TSH-producing and nonproducing transplantable tumors derived from murine thyrotroph cells, referred to as TtT-97 and MGH 101A, respectively, to identify nuclear factors which selectively interact with the mTSH beta gene. DNase I protection analyses demonstrate that factors present in TtT-97 nuclear extracts bind with high affinity to five separate sites in the TSH beta promoter region, denoted as distal D1 (-253 to -227) and proximal, P1 (-76 to -68), P2 (-106 to -98), P3 (-126 to -112), and P4 (-142 to -131) footprints. By contrast, non-TSH beta expressing thyrotroph cell nuclear extracts and L-cell nonpituitary cell extracts did not appear to footprint the D1 site; whereas the nonpituitary nuclear extracts revealed minimal DNase I protection in the P1-P4 regions. These data show that the distal D1 site is thyrotroph specific and contains a 6 base pair direct repeat sequence (5'-AGATAT-3'). Factor occupancy of the D1 site is protein dependent, occurs rapidly (less than 15 sec), is destabilized by 170 mM KCl, and results in an associated DNase I hypersensitive region. A double-stranded oligonucleotide spanning the D1 footprint competes only the distal factor binding region. Transfection of plasmid constructs containing progressive 5'-deletions of the mTSH beta promoter linked to the reporter gene luciferase into primary TtT-97 cells demonstrate a marked decrease in activity between the regions -270 and -79, which contains the D1 region.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Identification of thyrotroph-specific factors and cis-acting sequences of the murine thyrotropin beta subunit gene. 279 1

The insulin-like growth factor I (IGF-I) receptor mediates signal transduction by the IGFs and plays a critical role in growth and development. The proximal promoter region of the rat IGF-I receptor gene contains multiple Sp1 consensus-binding sites (GC boxes). Various promoter fragments fused to a luciferase reporter gene were transiently cotransfected together with an Sp1 expression vector into Drosophila Schneider cells, which lack endogenous Sp1. A proximal promoter fragment containing 476 nucleotides of 5'-flanking region and 640 nucleotides of 5'-untranslated region was strongly activated by Sp1 (an average of 116-fold), and progressive 5'-deletions of the promoter that sequentially removed GC boxes reduced Sp1 activation to 15-fold over basal promoter activity. DNase I footprinting studies with purified Sp1 protein revealed four GC boxes in the 5'-flanking region of the promoter and one homopurine/homopyrimidine motif (CT element) in the 5'-untranslated region that bound Sp1. Mutation of the CT element reduced Sp1 activation by 70%. Taken together, these results demonstrate that Sp1 can regulate expression of the IGF-I receptor promoter by acting both on GC boxes in the 5'-flanking region of the promoter and on a CT element in the 5'-untranslated region.
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PMID:Regulation of insulin-like growth factor I receptor gene expression by Sp1: physical and functional interactions of Sp1 at GC boxes and at a CT element. 749 Nov 7

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