scholarly journals Activation of proglucagon gene transcription through a novel promoter element by the caudal-related homeodomain protein cdx-2/3.

1996 ◽  
Vol 16 (1) ◽  
pp. 19-28 ◽  
Author(s):  
T Jin ◽  
D J Drucker
Keyword(s):  
Cell Types ◽  
Intestinal Cell ◽  
Homeodomain Protein ◽  
Promoter Element ◽  
Mobility Shift ◽  
Specific Expression ◽  
Coordinate Regulation ◽  
Cell Extracts ◽  
Homeobox Protein ◽  
Mobility Shift Assay

The proglucagon gene is expressed in a highly restricted tissue-specific manner in the A cells of the pancreatic islet and the L cells of the small and large intestines. The results of previous experiments indicate that cell-specific expression of the proglucagon gene is mediated by proteins that interact with the proximal G1 promoter element. We show here that the G1 element contains several AT-rich subdomains that bind proteins present in islet and enteroendocrine cell extracts. Electrophoretic mobility shift assay experiments using specific antisera identified the homeobox protein cdx-2/3 (which designates the same homeobox protein called cdx-2 for mice and cdx-3 for hamsters) as a major component of the G1-Gc2 complex in islet and intestinal cells. Mutations of the Gc element that decreased cdx-2/3 binding also resulted in decreased proglucagon promoter activity in islet and intestinal cell lines. The finding that cdx-2/3 mediates activation of the proglucagon promoter in both islet and enteroendocrine cells is consistent with the common endodermal lineage of these tissues and provides new insight into the coordinate regulation of genes expressed in both pancreatic and intestinal endocrine cell types.

scholarly journals An E-box-containing region is involved in the tissue-specific expression of the human MC2R gene

2004 ◽  
Vol 32 (3) ◽  
pp. 811-823 ◽  
Author(s):  
A Blondet ◽  
M Doghman ◽  
P Durand ◽  
M Begeot ◽  
D Naville
Keyword(s):  
Granulosa Cells ◽  
Gene Promoter ◽  
Cell Types ◽  
Melanocortin Receptor ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Specific Expression ◽  
E Box ◽  
Mobility Shift Assay

Expression of the melanocortin receptor (MC2R) gene is limited to adrenocortical cells and the aim of this study was to determine the factors responsible for this tissue specificity. We used different fragments of the human (h) MC2R gene promoter, inserted in a vector upstream of the luciferase reporter gene, to transiently transfect either bovine adrenocortical (BAC) cells or granulosa cells from bovine ovaries (B-Gran). Similar promoter activities were obtained in both cell types using constructs containing fragments up to 1017 bp of the hMC2R gene promoter. On the contrary, a 2-fold decrease was obtained after transfection of the B-Gran cells with vectors containing 1069 bp and more of the promoter. Results obtained here using BAC cells confirmed our previous data on human cells showing that steroidogenic factor 1 is the major transactivating factor involved in the basal expression of the hMC2R gene in adrenal cells. However, we showed that this factor did not permit, by itself, the expression of the hMC2R gene in B-Gran cells despite its expression in these cells. This study demonstrated for the first time that an E-box (located at -1020 bp) is involved in the repression of hMC2R gene expression in granulosa cells through interactions with several factors, such as activator protein 4, as suggested by electrophoretic mobility shift assay analyses.

Amifostine Inhibits Hematopoietic Progenitor Cell Apoptosis by Activating NF-κB/Rel Transcription Factors

Blood ◽  
1999 ◽  
Vol 94 (12) ◽  
pp. 4060-4066 ◽  
Author(s):  
Maria Fiammetta Romano ◽  
Annalisa Lamberti ◽  
Rita Bisogni ◽  
Corrado Garbi ◽  
Antonio M. Pagnano ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Cell Apoptosis ◽  
Progenitor Cell ◽  
Cell Types ◽  
Hematopoietic Progenitor Cell ◽  
Hematopoietic Progenitor ◽  
Mobility Shift ◽  
Human Cord Blood ◽  
Mobility Shift Assay ◽  
Induced Apoptosis

Abstract We investigated the involvement of NF-κB/Rel transcription factors that reportedly can inhibit apoptosis in various cell types in the antiapoptotic mechanism of the cytoprotectant amifostine. In the nontumorigenic murine myeloid progenitor 32D cells incubated with amifostine, we detected a reduction of the IκB cytoplasmic levels by Western blotting and a raising of nuclear NF-κB/Rel complexes by electrophoretic mobility shift assay. Amifostine inhibited by more than 30% the growth factor deprivation-induced apoptosis, whereas its effect failed when we blocked the NF-κB/Rel activity with an NF-κB/Rel-binding phosphorothioate decoy oligodeoxynucleotide. In human cord blood CD34+ cells, the NF-κB/Rel p65 subunit was detectable (using immunofluorescence analysis) mainly in the cytoplasm in the absence of amifostine, whereas its presence was appreciable in the nuclei of cells incubated with the cytoprotectant. In 4 CD34+ samples incubated for 3 days in cytokine-deficient conditions, cell apoptosis was reduced by more than 30% in the presence of amifostine (or amifostine plus a control oligo); the effect of amifostine was abolished in cultures with the decoy oligo. These findings indicate that the inhibition of hematopoietic progenitor cell apoptosis by amifostine requires the induction of NF-κB/Rel factors and that the latter can therefore exert an antiapoptotic activity in the hematopoietic progenitor cell compartment. Furthermore, the identification of this specific mechanism underlying the survival-promoting activity of amifostine lends support to the possible use of this agent in apoptosis-related pathologies, such as myelodysplasias.

Activation of octamer-containing promoters by either octamer-binding transcription factor 1 (OTF-1) or OTF-2 and requirement of an additional B-cell-specific component for optimal transcription of immunoglobulin promoters

1990 ◽  
Vol 10 (12) ◽  
pp. 6204-6215
Author(s):  
A Pierani ◽  
A Heguy ◽  
H Fujii ◽  
R G Roeder
Keyword(s):  
B Cell ◽  
Regulatory Element ◽  
Immunoglobulin Gene ◽  
Mobility Shift ◽  
Specific Expression ◽  
Tissue Specific ◽  
Activation Domains ◽  
Cell Extracts ◽  
Octamer Motif ◽  
High Level

Several distinct octamer-binding transcription factors (OTFs) interact with the sequence ATTTGCAT (the octamer motif), which acts as a transcription regulatory element for a variety of differentially controlled genes. The ubiquitous OTF-1 plays a role in expression of the cell cycle-regulated histone H2b gene as well as several other genes, while the tissue-specific OTF-2 has been implicated in the tissue-specific expression of immunoglobulin genes. In an attempt to understand the apparent transcriptional selectivity of these factors, we have investigated the physical and functional characteristics of OTF-1 purified from HeLa cells and both OTF-1 and OTF-2 purified from B cells. High-resolution footprinting and mobility shift-competition assays indicated that these factors were virtually indistinguishable in binding affinities and DNA-protein contacts on either the H2b or an immunoglobulin light-chain (kappa) promoter. In addition, each of the purified factors showed an equivalent intrinsic capacity to activate transcription from either immunoglobulin promoters (kappa and heavy chain) or the H2b promoter in OTF-depleted HeLa and B-cell extracts. However, with OTF-depleted HeLa extracts, neither factor could restore immunoglobulin gene transcription to the relatively high level observed in unfractionated B-cell extracts. Restoration of full immunoglobulin gene activity appears to require an additional B-cell regulatory component which interacts with the OTFs. The additional B-cell factor could act either by facilitating interaction of OTF activation domains with components of the general transcriptional machinery or by contributing a novel activation domain.

Olf-1-binding site: characterization of an olfactory neuron-specific promoter motif

1993 ◽  
Vol 13 (5) ◽  
pp. 3002-3014
Author(s):  
K Kudrycki ◽  
C Stein-Izsak ◽  
C Behn ◽  
M Grillo ◽  
R Akeson ◽  
...  
Keyword(s):  
Nuclear Protein ◽  
Consensus Sequence ◽  
Binding Motif ◽  
Sequence Motif ◽  
Olfactory Neuron ◽  
Sequence Motifs ◽  
Mobility Shift ◽  
Specific Expression ◽  
Mobility Shift Assay

We report characterization of several domains within the 5' flanking region of the olfactory marker protein (OMP) gene that may participate in regulating transcription of this and other olfactory neuron-specific genes. Analysis by electrophoretic mobility shift assay and DNase I footprinting identifies two regions that contain a novel sequence motif. Interactions between this motif and nuclear proteins were detected only with nuclear protein extracts derived from olfactory neuroepithelium, and this activity is more abundant in olfactory epithelium enriched in immature neurons. We have designated a factor(s) involved in this binding as Olf-1. The Olf-1-binding motif consensus sequence was defined as TCCCC(A/T)NGGAG. Studies with transgenic mice indicate that a 0.3-kb fragment of the OMP gene containing one Olf-1 motif is sufficient for olfactory tissue-specific expression of the reporter gene. Some of the other identified sequence motifs also interact specifically with olfactory nuclear protein extracts. We propose that Olf-1 is a novel, olfactory neuron-specific trans-acting factor involved in the cell-specific expression of OMP.

Identification of a killer cell-specific regulatory element of the mouse perforin gene: an Ets-binding site-homologous motif that interacts with Ets-related proteins

1993 ◽  
Vol 13 (11) ◽  
pp. 6690-6701
Author(s):  
H Koizumi ◽  
M F Horta ◽  
B S Youn ◽  
K C Fu ◽  
B S Kwon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Binding Site ◽  
Protein Interactions ◽  
Regulatory Element ◽  
Killer Cell ◽  
Mobility Shift ◽  
Specific Expression ◽  
Native Molecular Mass ◽  
Mobility Shift Assay

The gene encoding the cytolytic protein perforin is selectively expressed by activated killer lymphocytes. To understand the mechanisms underlying the cell-type-specific expression of this gene, we have characterized the regulatory functions and the DNA-protein interactions of the 5'-flanking region of the mouse perforin gene (Pfp). A region extending from residues +62 through -141, which possesses the essential promoter activity, and regions further upstream, which are able to either enhance or suppress gene expression, were identified. The region between residues -411 and -566 was chosen for further characterization, since it contains an enhancer-like activity. We have identified a 32-mer sequence (residues -491 to -522) which appeared to be capable of enhancing gene expression in a killer cell-specific manner. Within this segment, a 9-mer motif (5'-ACAGGAAGT-3', residues -505 to -497; designated NF-P motif), which is highly homologous to the Ets proto-oncoprotein-binding site, was found to interact with two proteins, NF-P1 and NF-P2. NF-P2 appears to be induced by reagents known to up-regulate the perforin message level and is present exclusively in killer cells. Electrophoretic mobility shift assay and UV cross-linking experiments revealed that NF-P1 and NF-P2 may possess common DNA-binding subunits. However, the larger native molecular mass of NF-P1 suggests that NF-P1 contains an additional non-DNA-binding subunit(s). In view of the homology between the NF-P motif and other Ets proto-oncoprotein-binding sites, it is postulated that NF-P1 and NF-P2 belong to the Ets protein family. Results obtained from the binding competition assay, nevertheless, suggest that NF-P1 and NF-P2 are related to but distinct from Ets proteins, e.g., Ets-1, Ets-2, and NF-AT/Elf-1, known to be expressed in T cells.

Abstract 447: Ac-SDKP Suppresses TNFα-induced ICAM-1 Expression In Endothelial Cells Via Inhibition Of IκB Kinase And NF-κB Activation

Hypertension ◽  
2014 ◽  
Vol 64 (suppl_1) ◽  
Author(s):  
Liping Zhu ◽  
Xiao-Ping Yang ◽  
Pablo Nakagawa ◽  
Nour-Eddine Rhaleb ◽  
Pamela Harding ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Types ◽  
Intercellular Adhesion Molecule ◽  
Intercellular Adhesion Molecule 1 ◽  
Human Coronary Artery ◽  
Mobility Shift ◽  
Autoimmune Myocarditis ◽  
Iκb Kinase ◽  
Subsequent Degradation ◽  
Mobility Shift Assay

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a naturally occurring tetrapeptide that prevents inflammation and fibrosis in hypertension and other cardiovascular diseases. We previously showed that Ac-SDKP decreased transcription factor NF-κB activation in angiotensin II-induced hypertension and also reduced intercellular adhesion molecule-1 (ICAM-1) expression in an experimental autoimmune myocarditis and hypertension animal model. However, the mechanisms by which Ac-SDKP down-regulated ICAM-1 expression are unclear. TNFα is a proinflammatory cytokine that induces ICAM-1 expression on different cell types. We hypothesized that Ac-SDKP suppresses TNFα-induced ICAM-1 via inhibition of IκB kinase (IKK) and subsequently by blockade of NF-κB activation. Human coronary artery endothelial cells were treated with Ac-SDKP or vehicle and then stimulated with TNFα (0.5 ng/ml). ICAM-1 protein expression and phosphorylation of IKK (p-IKK), inhibitory κB (p-IκB), p38 (p-p38) and ERK (p-ERK) were measured by Western Blot. Activation of NF-κB was determined by electrophoretic mobility shift assay (EMSA). ICAM-1 expression was virtually undetectable under basal conditions, but greatly increased by TNFα. Ac-SDKP dose-dependently suppressed TNFα-induced ICAM-1 expression (set at a value of 1.0 arbitrary units, AU) to 0.67±0.13 (p<0.05), 0.51±0.12 (p<0.01) and 0.39±0.09 AU (p<0.01) at 0.1 nM, 1 nM and 10 nM, respectively. In addition, Ac-SDKP (10 nM) inhibited TNFα-induced p-IKK from 1.0 to 0.71±0.02 AU (p<0.01). Ac-SDKP also inhibited TNFα-induced IKKβ expression from 1.0 to 0.64±0.12 AU (p<0.05), without affecting IKKα expression. Furthermore, Ac-SDKP inhibited TNFα-induced p-IκB from 1.0 to 0.54±0.03 AU (p<0.01). EMSA results showed that Ac-SDKP inhibited TNFα-mediated activation of NF-κB, which was 0.63±0.04-fold of TNFα-treated cells. However, Ac-SDKP had no effect on TNFα-induced p-p38 and p-ERK. Thus, we conclude that Ac-SDKP inhibits TNFα-induced IKK and subsequent degradation of IκB, thereby preventing NF-κB activation and ICAM-1 expression. These inhibitory effects are independent of p38 and ERK.

HnRNPU regulates intra- and intercellular microRNA trafficking in a sequence specific manner

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
A Zietzer ◽  
M.R Hosen ◽  
P.R Goody ◽  
N Werner ◽  
G Nickenig ◽  
...  
Keyword(s):  
Cardiac Fibroblasts ◽  
Cell Types ◽  
Underlying Mechanism ◽  
Negative Control ◽  
Funding Source ◽  
Mobility Shift ◽  
Pulldown Experiment ◽  
Mobility Shift Assay ◽  
Human Cardiac Fibroblasts ◽  
Plasmid Transfection

Abstract Introduction The transfer of microRNAs (miRs) through extracellular vesicles (EVs) is a well-established mechanism of vascular intercellular communication. In a previous study, we have identified the heterogeneous nuclear ribonucleoprotein U (hnRNPU) as a potent regulator of the vesicular export of a specific set of miRs in endothelial cells (ECs). Downregulation of hnRNPU caused an increased export of miRs into large EVs and lead to a reduction of the migratory capacity in EV up-taking ECs. The underlying mechanism, how hnRNPU recognizes only certain miRs and regulates their export, however, remained unclear. Methods and results Increasingly exported microRNAs into EVs after siRNA-mediated downregulation of hnRNPU were identified in a miR array and used to perform a sequence analysis with the Multiple Em for Motif Elicitation tool (MEME). The software identified the motif AAMRUGCU to be significantly enriched within the regulated miRs. Binding of hnRNPU to miR-30c-5p, which was one of the most significantly increased miRs in EVs upon downregulation of hnRNPU, was confirmed in an immunoprecipitation and an RNA-pulldown experiment. In order to investigate, if the in silico motif AARUGCU was the binding site of miR-30c-5p and hnRNPU an electrophoretic mobility shift assay (EMSA) was conducted with native miR-30c-5p, mutated miR-30c-5p as well as miR-125a-3p as a negative control. The EMSA showed that a partial mutation, which conserves the purine / pyrimidine structure in the motif of miR-30c-5p does not affect binding, while a complete disruption of the motif structure abolishes binding of hnRNPU and miR-30c-5p. Furthermore, we found that hnRNPU protects miR-30c-5p from rapid degradation after transcriptional blockage by Actinomycin D. Binding of miR-30c-5p to the predominantly nuclearly expressed hnRNPU retains miR-30c-5p in the nucleus. HnRNPU knockdown leads to a redistribution of miR-30c-5p from the nucleus to the cytoplasm. The regulating effect of hnRNPU expression on vesicular miR export was confirmed by overexpression of hnRNPU through plasmid transfection. In contrast to siRNA-mediated downregulation, overexpression of hnRNPU lead to a decrease of vesicular miR-30c-5p levels. In order to extent our findings to other cell types with high vascular relevance, siRNA mediated downregulation of hnRNPU was performed in human cardiac fibroblasts (HCF). Similar to our findings in ECs, downregulation of hnRNPU in HCFs lead to an increase in vesicular miR-30c-5p levels. Conclusion In conclusion, we show that hnRNPU binds miRs, which contain the motif AAMRUGCU. This leads to nuclear retainment, stabilization and inhibits vesicular export, which can be further decreased by overexpression of hnRNPU and enhanced by downregulation of hnRNPU. The effect is not restricted to ECs but applies also to HCFs. hnRNPU may therefore be an important target to influence intra- and intercellular trafficking of a specific set of miRs including miR-30c-5p. Funding Acknowledgement Type of funding source: Public hospital(s). Main funding source(s): Medical faculty of the University of Bonn, German Cardiac Society

scholarly journals The retinoblastoma gene product RB stimulates Sp1-mediated transcription by liberating Sp1 from a negative regulator.

1994 ◽  
Vol 14 (7) ◽  
pp. 4380-4389 ◽  
Author(s):  
L I Chen ◽  
T Nishinaka ◽  
K Kwan ◽  
I Kitabayashi ◽  
K Yokoyama ◽  
...  
Keyword(s):  
Dna Binding ◽  
Gene Product ◽  
Binding Activity ◽  
Negative Regulator ◽  
Control Element ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Dna Binding Activity ◽  
Cell Extracts ◽  
Mobility Shift Assay

Studies have demonstrated that the retinoblastoma susceptibility gene product, RB, can either positively or negatively regulate expression of several genes through cis-acting elements in a cell-type-dependent manner. The nucleotide sequence of the retinoblastoma control element (RCE) motif, GCCACC or CCACCC, and the Sp1 consensus binding sequence, CCGCCC, can confer equal responsiveness to RB. Here, we report that RB activates transcription of the c-jun gene through the Sp1-binding site within the c-jun promoter. Preincubation of crude nuclear extracts with monoclonal antibodies to RB results in reduction of Sp1 complexes in a mobility shift assay, while addition of recombinant RB in mobility shift assay mixtures with CCL64 cell extracts leads to an enhancement of DNA-binding activity of SP1. These results suggest that RB is directly or indirectly involved in Sp1-DNA binding activity. A mechanism by which RB regulates transactivation is indicated by our detection of a heat-labile and protease-sensitive Sp1 negative regulator(s) (Sp1-I) that specifically inhibits Sp1 binding to a c-jun Sp1 site. This inhibition is reversed by addition of recombinant RB proteins, suggesting that RB stimulates Sp1-mediated transactivation by liberating Sp1 from Sp1-I. Additional evidence for Sp1-I involvement in Sp1-mediated transactivation was demonstrated by cotransfection of RB, GAL4-Sp1, and a GAL4-responsive template into CV-1 cells. Finally, we have identified Sp1-I, a approximately 20-kDa protein(s) that inhibits the Sp1 complexes from binding to DNA and that is also an RB-associated protein. These findings provide evidence for a functional link between two distinct classes of oncoproteins, RB and c-Jun, that are involved in the control of cell growth, and also define a novel mechanism for the regulation of c-jun expression.

scholarly journals Olf-1-binding site: characterization of an olfactory neuron-specific promoter motif.

1993 ◽  
Vol 13 (5) ◽  
pp. 3002-3014 ◽  
Author(s):  
K Kudrycki ◽  
C Stein-Izsak ◽  
C Behn ◽  
M Grillo ◽  
R Akeson ◽  
...  
Keyword(s):  
Nuclear Protein ◽  
Consensus Sequence ◽  
Binding Motif ◽  
Sequence Motif ◽  
Olfactory Neuron ◽  
Sequence Motifs ◽  
Mobility Shift ◽  
Specific Expression ◽  
Mobility Shift Assay

We report characterization of several domains within the 5' flanking region of the olfactory marker protein (OMP) gene that may participate in regulating transcription of this and other olfactory neuron-specific genes. Analysis by electrophoretic mobility shift assay and DNase I footprinting identifies two regions that contain a novel sequence motif. Interactions between this motif and nuclear proteins were detected only with nuclear protein extracts derived from olfactory neuroepithelium, and this activity is more abundant in olfactory epithelium enriched in immature neurons. We have designated a factor(s) involved in this binding as Olf-1. The Olf-1-binding motif consensus sequence was defined as TCCCC(A/T)NGGAG. Studies with transgenic mice indicate that a 0.3-kb fragment of the OMP gene containing one Olf-1 motif is sufficient for olfactory tissue-specific expression of the reporter gene. Some of the other identified sequence motifs also interact specifically with olfactory nuclear protein extracts. We propose that Olf-1 is a novel, olfactory neuron-specific trans-acting factor involved in the cell-specific expression of OMP.

Amifostine Inhibits Hematopoietic Progenitor Cell Apoptosis by Activating NF-κB/Rel Transcription Factors

Blood ◽  
1999 ◽  
Vol 94 (12) ◽  
pp. 4060-4066 ◽  
Author(s):  
Maria Fiammetta Romano ◽  
Annalisa Lamberti ◽  
Rita Bisogni ◽  
Corrado Garbi ◽  
Antonio M. Pagnano ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Cell Apoptosis ◽  
Progenitor Cell ◽  
Cell Types ◽  
Hematopoietic Progenitor Cell ◽  
Hematopoietic Progenitor ◽  
Mobility Shift ◽  
Human Cord Blood ◽  
Mobility Shift Assay ◽  
Induced Apoptosis

We investigated the involvement of NF-κB/Rel transcription factors that reportedly can inhibit apoptosis in various cell types in the antiapoptotic mechanism of the cytoprotectant amifostine. In the nontumorigenic murine myeloid progenitor 32D cells incubated with amifostine, we detected a reduction of the IκB cytoplasmic levels by Western blotting and a raising of nuclear NF-κB/Rel complexes by electrophoretic mobility shift assay. Amifostine inhibited by more than 30% the growth factor deprivation-induced apoptosis, whereas its effect failed when we blocked the NF-κB/Rel activity with an NF-κB/Rel-binding phosphorothioate decoy oligodeoxynucleotide. In human cord blood CD34+ cells, the NF-κB/Rel p65 subunit was detectable (using immunofluorescence analysis) mainly in the cytoplasm in the absence of amifostine, whereas its presence was appreciable in the nuclei of cells incubated with the cytoprotectant. In 4 CD34+ samples incubated for 3 days in cytokine-deficient conditions, cell apoptosis was reduced by more than 30% in the presence of amifostine (or amifostine plus a control oligo); the effect of amifostine was abolished in cultures with the decoy oligo. These findings indicate that the inhibition of hematopoietic progenitor cell apoptosis by amifostine requires the induction of NF-κB/Rel factors and that the latter can therefore exert an antiapoptotic activity in the hematopoietic progenitor cell compartment. Furthermore, the identification of this specific mechanism underlying the survival-promoting activity of amifostine lends support to the possible use of this agent in apoptosis-related pathologies, such as myelodysplasias.

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