Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Transcriptional activation of human 12-lipoxygenase gene promoter is mediated through Sp1 consensus sites in A431 cells.

ABSTRACT: The functional 5' flanking region of the human 12-lipoxygenase in epidermoid carcinoma A431 cells was characterized. By a primer extension method, the transcription initiation sites were mapped at -47 adenosine, -48 guanosine and -55 guanosine upstream of the ATG translation start codon. Transient transfection with a series of 5' and 3' deletion constructs showed that the 5' flanking region spanning from -224 to -100 bp was important for the basal expression of 12-lipoxygenase gene. Gel mobility shift assays with antibodies of transcription factors showed that both Sp1 and Sp3 required highly GC-rich Sp1 sites within this region for binding. Disruption of two Sp1 recognition motifs residing at -158 to -150 bp and -123 to -114 bp by site-directed mutagenesis markedly reduced the basal 12-lipoxygenase promoter activity and abolished the retarded bands in a gel-shift assay, indicating that these two Sp1-binding sites were essential for gene expression. The same two Sp1-binding sites in this promoter region were also responsible for epidermal growth factor (EGF)-induced expression of 12-lipoxygenase gene. Moreover, EGF also induced the transcriptional activation of luciferase driven by SV40 early promoter, which contained rich Sp1-binding sites. Taken together, the results suggest that two specific Sp1 consensus sites are involved in the mediation of the basal promoter activity as well as EGF induction of the 12-lipoxygenase gene and that Sp1 and Sp3 transcription factors might have a role in their regulation.

SUBMITTER: Liu YW

PROVIDER: S-EPMC1218409 | biostudies-other | 1997 May

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Transcriptional induction of DLC-1 gene through Sp1 sites by histone deacetylase inhibitors in gastric cancer cells.

Project description:We previously reported that trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, induced DLC-1 mRNA expression and accumulated acetylated histones H3 and H4 associated with the DLC-1 promoter in DLC-1 non-expressing gastric cancer cells. In this study, we demonstrated the molecular mechanisms by which TSA induced the DLC-1 gene expression. Treatment of the gastric cancer cells with TSA activates the DLC-1 promoter activity through Sp1 sites located at -219 and -174 relative to the transcription start site. Electrophoretic mobility-shift assay (EMSA) revealed that Sp1 and Sp3 specifically interact with these Sp1 sites and showed that TSA did not change their binding activities. The ectopic expression of Sp1, but not Sp3, enhances the DLC-1 promoter responsiveness by TSA. Furthermore, the TSA-induced DLC-1 promoter activity was increased by p300 expression and reduced by knockdown of p300. These results demonstrated the requirement of specific Sp1 sites and dependence of Sp1 and p300 for TSA-mediated activation of DLC-1 promoter.

| S-EPMC2679347 | biostudies-literature

Promoter-mediated transcriptional dynamics.

Project description:Genes in eukaryotic cells are typically regulated by complex promoters containing multiple binding sites for a variety of transcription factors, but how promoter dynamics affect transcriptional dynamics has remained poorly understood. In this study, we analyze gene models at the transcriptional regulation level, which incorporate the complexity of promoter structure (PS) defined as transcriptional exits (i.e., ON states of the promoter) and the transition pattern (described by a matrix consisting of transition rates among promoter activity states). We show that multiple exits of transcription are the essential origin of generating multimodal distributions of mRNA, but promoters with the same transition pattern can lead to multimodality of different modes, depending on the regulation of transcriptional factors. In turn, for similar mRNA distributions in the models, the mean ON or OFF time distributions may exhibit different characteristics, thus providing the supplemental information on PS. In addition, we demonstrate that the transcriptional noise can be characterized by a nonlinear function of mean ON and OFF times. These results not only reveal essential characteristics of promoter-mediated transcriptional dynamics but also provide signatures useful for inferring PS based on characteristics of transcriptional outputs.

| S-EPMC3907263 | biostudies-literature

G-Quadruplex Modulation of SP1 Functional Binding Sites at the KIT Proximal Promoter.

Project description:The regulation of conformational arrangements of gene promoters is a physiological mechanism that has been associated with the fine control of gene expression. Indeed, it can drive the time and the location for the selective recruitment of proteins of the transcriptional machinery. Here, we address this issue at the KIT proximal promoter where three G-quadruplex forming sites are present (kit1, kit2 and kit*). On this model, we focused on the interplay between G-quadruplex (G4) formation and SP1 recruitment. By site directed mutagenesis, we prepared a library of plasmids containing mutated sequences of the WT KIT promoter that systematically exploited different G4 formation attitudes and SP1 binding properties. Our transfection data showed that the three different G4 sites of the KIT promoter impact on SP1 binding and protein expression at different levels. Notably, kit2 and kit* structural features represent an on-off system for KIT expression through the recruitment of transcription factors. The use of two G4 binders further helps to address kit2-kit* as a reliable target for pharmacological intervention.

| S-EPMC7795597 | biostudies-literature

Protein kinase C regulation of 12-lipoxygenase-mediated human platelet activation.

Project description:Platelet activation is important in the regulation of hemostasis and thrombosis. Uncontrolled activation of platelets may lead to arterial thrombosis, which is a major cause of myocardial infarction and stroke. After activation, metabolism of arachidonic acid (AA) by 12-lipoxygenase (12-LOX) may play a significant role in regulating the degree and stability of platelet activation because inhibition of 12-LOX significantly attenuates platelet aggregation in response to various agonists. Protein kinase C (PKC) activation is also known to be an important regulator of platelet activity. Using a newly developed selective inhibitor for 12-LOX and a pan-PKC inhibitor, we investigated the role of PKC in 12-LOX-mediated regulation of agonist signaling in the platelet. To determine the role of PKC within the 12-LOX pathway, a number of biochemical endpoints were measured, including platelet aggregation, calcium mobilization, and integrin activation. Inhibition of 12-LOX or PKC resulted in inhibition of dense granule secretion and attenuation of both aggregation and αIIbβ(3) activation. However, activation of PKC downstream of 12-LOX inhibition rescued agonist-induced aggregation and integrin activation. Furthermore, inhibition of 12-LOX had no effect on PKC-mediated aggregation, indicating that 12-LOX is upstream of PKC. These studies support an essential role for PKC downstream of 12-LOX activation in human platelets and suggest 12-LOX as a possible target for antiplatelet therapy.

| S-EPMC3286293 | biostudies-literature

Characterization of human involucrin promoter distal regulatory region transcriptional activator elements-a role for Sp1 and AP1 binding sites.

Project description:Human involucrin (hINV) is an important precursor of the keratinocyte cornified envelope that is specifically expressed in the suprabasal layers of stratifying epithelia. Previous truncation and mutagenesis experiments have shown that an activator protein 1 (Ap1) site, AP1-5, located 2100bp upstream of the transcription start site, is required for optimal promoter activity. These previous studies suggest that AP1-5 is part of a distal regulatory region spanning nucleotides -2473 to -2088. In the present report, we study the distal regulatory region (DRR), which surrounds AP1-5. Our studies show that this region contains weak and strong activator elements spanning nucleotides -2473/-2216 and -2140/-2088, respectively. The strong activator element contains AP1-5 and an adjacent specificity protein 1 (Sp1) site. The AP1-5 site is absolutely required for DRR activity, as its mutation reduces transcription to basal levels. Mutagenesis studies of the AP1-5 and Sp1 sites in the presence or absence of the weak activator element indicate that the Sp1 site and the weak activator element synergistically activate the AP1-5 site-dependent transcription. The cooperation between the Sp1 and AP1-5 sites is also observed in the context of the full-length promoter. Gel mobility shift and supershift studies show that Sp1, but not Sp2, Sp3 or Sp4 binds to the Sp1 site. When the Sp1 site is mutated or the distance between the AP1-5 and Sp1 site is increased, the binding of AP1 factors to AP1-5 is markedly reduced. Surprisingly, gel shift studies suggest that activation does not require the formation of a stable AP1/Sp1/DNA ternary complex. These studies suggest that the AP1-5 site is absolutely required for transcriptional activation, that the weak activator element and Sp1 sites serve to enhance this activation, and that the Sp1 site is required for optimal AP1 factor binding at the AP1-5 site.

| S-EPMC1219321 | biostudies-other

Characterization of a human 12/15-lipoxygenase promoter variant associated with atherosclerosis identifies vimentin as a promoter binding protein.

Project description:BackgroundSequence variation in the human 12/15 lipoxygenase (ALOX15) has been associated with atherosclerotic disease. We functionally characterized an ALOX15 promoter polymorphism, rs2255888, previously associated with carotid plaque burden.Methodology/principal findingsWe demonstrate specific in vitro and in vivo binding of the cytoskeletal protein, vimentin, to the ALOX15 promoter. We show that the two promoter haplotypes carrying alternate alleles at rs2255888 exhibit significant differences in promoter activity by luciferase reporter assay in two cell lines. Differences in in-vitro vimentin-binding to and formation of DNA secondary structures in the polymorphic promoter sequence are also detected by electrophoretic mobility shift assay and biophysical analysis, respectively. We show regulation of ALOX15 protein by vimentin.Conclusions/significanceThis study suggests that vimentin binds the ALOX15 promoter and regulates its promoter activity and protein expression. Sequence variation that results in changes in DNA conformation and vimentin binding to the promoter may be relevant to ALOX15 gene regulation.

| S-EPMC3413658 | biostudies-literature

12/15-Lipoxygenase-mediated enzymatic lipid oxidation regulates DC maturation and function.

Project description:DCs are able to undergo rapid maturation, which subsequently allows them to initiate and orchestrate T cell-driven immune responses. DC maturation must be tightly controlled in order to avoid random T cell activation and development of autoimmunity. Here, we determined that 12/15-lipoxygenase-meditated (12/15-LO-mediated) enzymatic lipid oxidation regulates DC activation and fine-tunes consecutive T cell responses. Specifically, 12/15-LO activity determined the DC activation threshold via generation of phospholipid oxidation products that induced an antioxidative response dependent on the transcription factor NRF2. Deletion of the 12/15-LO-encoding gene or pharmacologic inhibition of 12/15-LO in murine or human DCs accelerated maturation and shifted the cytokine profile, thereby favoring the differentiation of Th17 cells. Exposure of 12/15-LO-deficient DCs to 12/15-LO-derived oxidized phospholipids attenuated both DC activation and the development of Th17 cells. Analysis of lymphatic tissues from 12/15-LO-deficient mice confirmed enhanced maturation of DCs as well as an increased differentiation of Th17 cells. Moreover, experimental autoimmune encephalomyelitis in mice lacking 12/15-LO resulted in an exacerbated Th17-driven autoimmune disease. Together, our data reveal that 12/15-LO controls maturation of DCs and implicate enzymatic lipid oxidation in shaping the adaptive immune response.

| S-EPMC4463199 | biostudies-literature

Increased expression of colonic Wnt9A through Sp1-mediated transcriptional effects involving arylsulfatase B, chondroitin 4-sulfate, and galectin-3.

Project description:In cultured human colonic epithelial cells and mouse colonic tissue, exposure to the common food additive carrageenan leads to inflammation, activation of Wnt signaling, increased Wnt9A expression, and decline in the activity of the enzyme arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase). In this study, the novel transcriptional mechanism by which carrageenan and decline in ARSB increase Wnt9A expression in NCM460 and HT-29 human colonic epithelial cells and in mouse colon is presented. Increased expression of Wnt9A has been associated with multiple malignancies, including colon carcinoma, and with ectodermal and mesoendodermal morphogenesis. When ARSB activity was reduced by siRNA or by exposure to carrageenan (1 ?g/ml for 24 h), degradation of chondroitin 4-sulfate (C4S) was inhibited, leading to accumulation of more highly sulfated C4S, which binds less galectin-3, a ?-galactoside-binding protein. Nuclear galectin-3 increased and mediated increased binding of Sp1 to the Sp1 consensus sequence in the Wnt9A promoter, shown by oligonucleotide-binding assay and by chromatin immunoprecipitation assay. When galectin-3 was silenced, the increases in Sp1 binding to the Wnt9A promoter and in Wnt9A expression, which followed carrageenan or ARSB silencing, were inhibited. Mithramycin A, a specific inhibitor of Sp1 oligonucleotide binding, and Sp1 siRNA blocked the carrageenan- and ARSB siRNA-induced increases in Wnt9A expression. These studies reveal how carrageenan exposure can lead to transcriptional events in colonic epithelial cells through decline in arylsulfatase B activity, with subsequent impact on C4S, galectin-3, Sp1, and Wnt9A and can exert significant effects on Wnt-initiated signaling and related vital cell processes.

| S-EPMC4067192 | biostudies-literature

Molecular and functional characterization of the promoter region of the mouse LDH/C gene: enhancer-assisted, Sp1-mediated transcriptional activation.

Project description:Molecular and functional studies of the LDH/C 5' upstream promoter elements were undertaken to elucidate the molecular mechanisms involved in temporal activation of LDH/C gene expression in differentiating germ cells. Ligation mediated-PCR (LM-PCR) gene walking techniques were exploited to isolate a 2.1 kb fragment of the mouse LDH/C 5' promoter region. DNA sequence analysis of this isolated genomic fragment indicated that the mouse LDH/C promoter contained TATA and CCAT boxes as well as a GC-box (Sp1-binding site) situated upstream from the transcription start site. PCR-based in vivo DNase I footprinting analysis of a 600 bp fragment of the proximal LDH/C promoter region (-524/+38) in isolated mouse pachytene spermatocytes identified a single footprint over the GC-box motif. Three DNase I hypersensitive sites were also detectable in vivo, in a region containing (CT)n(GA)n repeats upstream from the CCAT box domain. Functional characterization of the promoter region was carried out in a rat C6 glioma cell line and an SV40 transformed germ cell line (GC-1 spg) using wild-type and mutated LDH/C promoter CAT reporter constructs. These studies provide experimental evidence suggesting that transcriptional activation of the LDH/C promoter is regulated by enhancer-mediated coactivation of the Sp1 proteins bound to the GC-box motif footprinted in vivo in pachytene spermatocytes.

| S-EPMC146730 | biostudies-other

12-lipoxygenase activity plays an important role in PAR4 and GPVI-mediated platelet reactivity.

Project description:Following initial platelet activation, arachidonic acid is metabolised by cyclooxygenase-1 and 12-lipoxygenase (12-LOX). While the role of 12-LOX in the platelet is not well defined, recent evidence suggests that it may be important for regulation of platelet activity and is agonist-specific in the manner in which it regulates platelet function. Using small molecule inhibitors selective for 12-LOX and 12-LOX-deficient mice, the role of 12-LOX in regulation of human platelet activation and thrombosis was investigated. Pharmacologically inhibiting 12-LOX resulted in attenuation of platelet aggregation, selective inhibition of dense versus alpha granule secretion, and inhibition of platelet adhesion under flow for PAR4 and collagen. Additionally, 12-LOX-deficient mice showed attenuated integrin activity to PAR4-AP and convulxin compared to wild-type mice. Finally, platelet activation by PARs was shown to be differentially dependent on COX-1 and 12-LOX with PAR1 relying on COX-1 oxidation of arachidonic acid while PAR4 being more dependent on 12-LOX for normal platelet function. These studies demonstrate an important role for 12-LOX in regulating platelet activation and thrombosis. Furthermore, the data presented here provide a basis for potentially targeting 12-LOX as a means to attenuate unwanted platelet activation and clot formation.

| S-EPMC3960303 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data