scholarly journals Interplay between E-box and NF-κB in Regulation of A20 Gene by DRB Sensitivity-inducing Factor (DSIF)

2007 ◽  
Vol 283 (3) ◽  
pp. 1317-1323 ◽  
Author(s):  
Liat Amir-Zilberstein ◽  
Rivka Dikstein
Keyword(s):  
Protein Complexes ◽  
Core Promoter ◽  
Elongation Factor ◽  
Mobility Shift ◽  
Dynamic Regulation ◽  
Upstream Promoter ◽  
E Box ◽  
Electrophoretic Mobility Shift Assays ◽  
Specific Control ◽  
A20 Gene

The NF-κB target gene A20 serves as a paradigm for gene-specific control of transcription elongation. This gene is regulated by the elongation factor DSIF (DRB sensitivity-inducing factor) under basal and NF-κB-activated states by two distinct mechanisms. Prior to NF-κB stimulation, the A20 gene is occupied by polymerase II, and elongation is inhibited by DSIF. This inhibition is mediated by an upstream promoter element termed ELIE (elongation inhibitory element). Upon NF-κB activation, inhibition of the A20 gene by DSIF persists, but now NF-κB and the core promoter regulate DSIF instead of ELIE. Here we investigated the regulation of DSIF by ELIE and the regulatory switch from ELIE to NF-κB following NF-κB induction. Electrophoretic mobility shift assays revealed two distinct protein complexes that specifically interact with ELIE, one of which is the E-box protein USF1. Interestingly, USF1 is displaced from the A20 promoter upon induction of NF-κB. A mutation in the E-box section of ELIE diminished the binding of USF1 and DSIF recruitment. Consistent with these findings, the E-box is crucial for DSIF inhibition in resting, but not NF-κB-stimulated, cells. These findings reveal a dynamic regulation of DSIF involving either E-box or NF-κB depending on the physiological circumstances.

scholarly journals Regulation of the murine alpha B-crystallin/small heat shock protein gene in cardiac muscle.

1995 ◽  
Vol 15 (12) ◽  
pp. 7081-7090 ◽  
Author(s):  
R Gopal-Srivastava ◽  
J I Haynes ◽  
J Piatigorsky
Keyword(s):  
Skeletal Muscle ◽  
Heat Shock ◽  
Serum Response Factor ◽  
Small Heat Shock Protein ◽  
Mobility Shift ◽  
Enhancer Activity ◽  
E Box ◽  
Carg Box ◽  
Electrophoretic Mobility Shift Assays ◽  
Serum Response

The murine alpha B-crystallin/small heat shock protein gene is expressed at high levels in the lens and at lower levels in the heart, skeletal muscle, and numerous other tissues. Previously we have found a skeletal-muscle-preferred enhancer at positions -427 to -259 of the alpha B-crystallin gene containing at least four cis-acting regulatory elements (alpha BE-1, alpha BE-2, alpha BE-3, and MRF, which has an E box). Here we show that in transgenic mice, the alpha B-crystallin enhancer directs the chloramphenicol acetyltransferase reporter gene driven by the alpha B-crystallin promoter specifically to myocardiocytes of the heart. The alpha B-crystallin enhancer was active in conjugation with the herpes simplex virus thymidine kinase promoter/human growth hormone reporter gene in transfected rat myocardiocytes. DNase I footprinting and site-specific mutagenesis experiments showed that alpha BE-1, alpha BE-2, alpha BE-3, MRF, and a novel, heart-specific element called alpha BE-4 are required for alpha B-crystallin enhancer activity in transfected myocardiocytes. By contrast, alpha BE-4 is not utilized for enhancer activity in transfected lens or skeletal muscle cell lines. Alpha BE-4 contains an overlapping heat shock sequence and a reverse CArG box [5'-GG(A/T)6CC-3']. Electrophoretic mobility shift assays with an antibody to serum response factor and a CArG-box-competing sequence from the c-fos promoter indicated that a cardiac-specific protein with DNA-binding and antigenic similarities to serum response factor binds to alpha BE-4 via the reverse CArG box; electrophoretic mobility shift assays and antibody experiments with anti-USF antiserum and heart nuclear extract also raised the possibility that the MRF E box utilizes USF or an antigenically related protein. We conclude that the activity of the alpha B-crystallin enhancer in the heart utilizes a reverse CArG box and an E-box-dependent pathway.

Myogenic regulatory factors transactivate the Tceal7 gene and modulate muscle differentiation

2010 ◽  
Vol 428 (2) ◽  
pp. 213-221 ◽  
Author(s):  
Xiaozhong Shi ◽  
Daniel J. Garry
Keyword(s):  
Skeletal Muscle ◽  
Muscle Regeneration ◽  
Muscle Development ◽  
Cellular Proliferation ◽  
Elongation Factor ◽  
Muscle Differentiation ◽  
Myogenic Regulatory Factors ◽  
Mobility Shift ◽  
Regulatory Factors ◽  
E Box

Recurrent injuries eventually exhaust the capacity of skeletal muscle to fully restore or regenerate its cellular architecture. Therefore a comprehensive understanding of the muscle regeneration programme is needed to provide a platform for new therapies for devastating diseases such as Duchenne muscular dystrophy. To begin to decipher the molecular programme that directs muscle regeneration, we undertook an unbiased strategy using microarray analysis of cardiotoxin-injured skeletal muscle at defined time periods in the adult mouse. Using this strategy, we identified Tceal7 [transcription elongation factor A (SII)-like 7], which was dynamically regulated during muscle regeneration. Our studies revealed that Tceal7 was restricted to the skeletal muscle lineage during embryogenesis. Using transgenic technologies and transcriptional assays, we defined an upstream 0.7 kb fragment of the Tceal7 gene that directed the LacZ reporter to the developing skeletal muscle lineage. Analysis of the Tceal7 promoter revealed evolutionarily conserved E-box motifs within the 0.7 kb upstream fragment that were essential for promoter activity, as mutation of the E-box motifs resulted in the loss of reporter expression in the somites of transgenic embryos. Furthermore, we demonstrated that MRFs (myogenic regulatory factors) were Tceal7 upstream transactivators using transcriptional assays, EMSAs (electrophoretic mobility-shift assays), and ChIP (chromatin immunoprecipitation) assays. Overexpression of Tceal7 in C2C12 myoblasts decreased cellular proliferation and enhanced differentiation. Further studies revealed that p27 expression was up-regulated following Tceal7 overexpression. These studies support the hypothesis that MRFs transactivate Tceal7 gene expression and promote muscle differentiation during muscle development and regeneration.

scholarly journals Transcription factor organic cation transporter 1 (OCT-1) affects the expression of porcine Klotho (KL) gene

2016 ◽  
Author(s):  
Yan Li ◽  
Lei Wang ◽  
Jiawei Zhou ◽  
Fenge Li
Keyword(s):  
Transcription Factor ◽  
Organic Cation ◽  
Core Promoter ◽  
Sequence Similarity ◽  
Organic Cation Transporter ◽  
Mobility Shift ◽  
Fragment Analysis ◽  
Intron 1 ◽  
Organic Cation Transporter 1 ◽  
Electrophoretic Mobility Shift Assays

Klotho (KL), originally discovered as an aging suppressor, was a membrane protein that shared sequence similarity with the β-glucosidase enzymes. Recent reports showed Klotho might have a role in adipocyte maturation and systemic glucose metabolism. However, little is known about the transcription factors involved in regulating the expression of porcine KL gene. Deletion fragment analysis identified KL-D2 (-418 bp to -3 bp) as the porcine KL core promoter. MARC0022311 in KL intron 1 appeared a polymorphism (A or G) in Landrace × DIV pigs, and relative luciferase activity of pGL3-D2-G was significantly higher than pGL3-D2-A. This was possibly the result of a change in KL binding ability with transcription factor organic cation transporter 1 (OCT-1), which was confirmed using electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP). Moreover, OCT-1 regulated endogenous KL expression by RNA interference. Our study indicates SNP MARC0022311 affects porcine KL expression by regulating its promoter activity via OCT-1.

Transcription factor AP-4 is a ligand for immunoglobulin-κ promoter E-box elements

2001 ◽  
Vol 354 (2) ◽  
pp. 431-438 ◽  
Author(s):  
Alaitz ARANBURU ◽  
Robert CARLSSON ◽  
Christine PERSSON ◽  
Tomas LEANDERSON
Keyword(s):  
Transcription Factor ◽  
Gene Families ◽  
Sequence Motifs ◽  
Mobility Shift ◽  
Conserved Sequence ◽  
Nuclear Extracts ◽  
E Box ◽  
Variable Gene ◽  
E Boxes ◽  
Electrophoretic Mobility Shift Assays

Immunoglobulin (Ig)-κ promoters from humans and mice share conserved sequences. The octamer element is common to all Ig promoters and pivotal for their function. However, other conserved sequence motifs, that differ between Ig variable gene families, are required for normal promoter function. These conserved motifs do not stimulate transcription in the absence of an octamer. One example is an E-box of the E47/E12 type (5′-CAGCTG-3′), which is found in all promoters of the human and murine Ig-κ gene subgroups/families, with the exception of subgroups II and VI and their related murine families. In the present study we show that the ubiquitously expressed transcription factor AP-4, and not E47, interacts specifically with the κ promoter E-boxes when tested in electrophoretic mobility-shift assays using nuclear extracts derived from human and murine B-cell lines. Furthermore, AP-4, unlike E47, did not act as a transactivator, which is in agreement with previous studies on intact κ promoters, showing that transcription is absent when the octamer element has been mutated. Based on these data, and the conservation of the 5′-CAGCTG-3′ motif among human and murine κ promoters, we propose that AP-4 is the major ligand for Ig-κ promoter E-boxes.

scholarly journals Regulation of the Human Proliferating Cell Nuclear Antigen Promoter by the Adenovirus E1A-Associated Protein p107

1998 ◽  
Vol 72 (2) ◽  
pp. 1138-1145 ◽  
Author(s):  
Benjamin H. Lee ◽  
Mingsong Liu ◽  
Michael B. Mathews
Keyword(s):  
Proliferating Cell Nuclear Antigen ◽  
Transcriptional Repression ◽  
Mutational Analysis ◽  
Protein Complexes ◽  
Nuclear Antigen ◽  
Mobility Shift ◽  
Adenovirus E1a ◽  
Nuclear Extracts ◽  
Electrophoretic Mobility Shift Assays ◽  
Proliferating Cell

ABSTRACT The adenovirus E1A 243R oncoprotein is capable of transactivating the expression of the human proliferating cell nuclear antigen (PCNA) promoter. Mutational analysis of the E1A 243R protein suggested that both its p300/CBP- and p107-binding regions are required for optimal induction of the PCNA promoter (C. Kannabiran, G. F. Morris, C. Labrie, and M. B. Mathews, J. Virol. 67:425–437, 1993). We show that overexpression of p107 antagonizes the induction of PCNA by E1A 243R in transient expression assays. This inhibition is largely independent of p107’s ability to interact with E1A 243R, because p107 mutants unable to bind to E1A 243R retain the ability to repress the E1A-activated PCNA promoter. Electrophoretic mobility shift assays with the PCNA promoter detected the presence of p107 in one of the major DNA-protein complexes, EH1, formed with HeLa cell nuclear extracts. Promoter mutations that disrupt the formation of complex EH1 abrogated p107’s ability to reverse E1A 243R-induced PCNA expression. The same mutations characterize a sequence important for the binding of transcription factor RFX1 (C. Labrie, G. F. Morris, and M. B. Mathews, Nucleic Acids Res. 23:3732–3741, 1995), implying that p107 antagonizes E1A 243R-induced PCNA expression through this RFX1-binding site. Our data are suggestive of a novel cooperative mechanism for transactivation of PCNA expression, in which E1A 243R relieves transcriptional repression exerted by p107 on the promoter.

scholarly journals Transcription factor organic cation transporter 1 (OCT-1) affects the expression of porcine Klotho (KL) gene

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2186 ◽  
Author(s):  
Yan Li ◽  
Lei Wang ◽  
Jiawei Zhou ◽  
Fenge Li
Keyword(s):  
Transcription Factor ◽  
Organic Cation ◽  
Core Promoter ◽  
Sequence Similarity ◽  
Organic Cation Transporter ◽  
Mobility Shift ◽  
Intron 1 ◽  
Organic Cation Transporter 1 ◽  
Immune Precipitation ◽  
Electrophoretic Mobility Shift Assays

Klotho (KL), originally discovered as an aging suppressor, is a membrane protein that shares sequence similarity with theβ-glucosidase enzymes. Recent reports showed Klotho might play a role in adipocyte maturation and systemic glucose metabolism. However, little is known about the transcription factors involved in regulating the expression of porcineKLgene. Deletion fragment analysis identified KL-D2 (−418 bp to −3 bp) as the porcineKLcore promoter. MARC0022311SNP (A or G) in KL intron 1 was detected in Landrace × DIV pigs using the Porcine SNP60 BeadChip. The pGL-D2-A and pGL-D2-G were constructed with KL-D2 and the intron fragment of different alleles and relative luciferase activity of pGL3-D2-G was significantly higher than that of pGL3-D2-A in the PK cells and ST cells. This was possibly the result of a change inKLbinding ability with transcription factor organic cation transporter 1 (OCT-1), which was confirmed using electrophoretic mobility shift assays (EMSA) and chromatin immune-precipitation (ChIP). Moreover, OCT-1 regulated endogenousKLexpression by RNA interference experiments. Our study indicates SNP MARC0022311 affects porcineKLexpression by regulating its promoter activity via OCT-1.

scholarly journals Transcription factor organic cation transporter 1 (OCT-1) affects the expression of porcine Klotho (KL) gene

2016 ◽  
Author(s):  
Yan Li ◽  
Lei Wang ◽  
Jiawei Zhou ◽  
Fenge Li
Keyword(s):  
Transcription Factor ◽  
Organic Cation ◽  
Core Promoter ◽  
Sequence Similarity ◽  
Organic Cation Transporter ◽  
Mobility Shift ◽  
Fragment Analysis ◽  
Intron 1 ◽  
Organic Cation Transporter 1 ◽  
Electrophoretic Mobility Shift Assays

Klotho (KL), originally discovered as an aging suppressor, was a membrane protein that shared sequence similarity with the β-glucosidase enzymes. Recent reports showed Klotho might have a role in adipocyte maturation and systemic glucose metabolism. However, little is known about the transcription factors involved in regulating the expression of porcine KL gene. Deletion fragment analysis identified KL-D2 (-418 bp to -3 bp) as the porcine KL core promoter. MARC0022311 in KL intron 1 appeared a polymorphism (A or G) in Landrace × DIV pigs, and relative luciferase activity of pGL3-D2-G was significantly higher than pGL3-D2-A. This was possibly the result of a change in KL binding ability with transcription factor organic cation transporter 1 (OCT-1), which was confirmed using electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP). Moreover, OCT-1 regulated endogenous KL expression by RNA interference. Our study indicates SNP MARC0022311 affects porcine KL expression by regulating its promoter activity via OCT-1.

Antiparasitic Treatment of Patients with P. falciparum Malaria Reduces the Ability of Patient Serum to Induce Tissue Factor by Decreasing NF-κB Activation

1995 ◽  
Vol 73 (01) ◽  
pp. 039-048 ◽  
Author(s):  
A Bierhaus ◽  
Ch J Hemmer ◽  
N Mackman ◽  
R Kutob ◽  
R Ziegler ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Falciparum Malaria ◽  
De Novo ◽  
Neutralizing Antibody ◽  
Mobility Shift ◽  
Before And After ◽  
Tf Gene ◽  
Electrophoretic Mobility Shift Assays ◽  
Antiparasitic Treatment ◽  
Stimulation Of

SummarySerum from patients with P. falciparum malaria at day 1 (pretherapy) induces tissue factor (TF) in cultured endothelial cells. TF induction depends on de novo transcription as shown in Nuclear Run On assays. Electrophoretic mobility shift assays demonstrated binding of AP-1 and NF- κB/Rel proteins to their recognition sites in the TF promotor. After therapy (day 28), stimulation of TF antigen by patient serum is reduced by 70%. When serum obtained before and after therapy was compared, a decrease of NF-κB activation was evident. Activation of NF-κB-like proteins was in part dependent on TNFα in patient serum, since a TNFα neutralizing antibody reduced induction of TF transcription and translation and induction of NF-κB-like proteins. Induction of TF activity was suppressed by pDTC, an inhibitor of NF-κB activation. When different promotor constructs of the TF gene were tested, induction was dependent upon the presence of the intact NF-κB-like binding site in the TF promotor. A mutant with deleted NF-κB, but intact AP-1 sites was not inducible. Mutation of the AP-1 sites did not prevent induction, but reduced inducibility by pretherapy serum. Therefore, NF-κB/Rel proteins are responsible for induction of TF transcription by pretherapy serum, but AP-1 is needed for highest inducibility. The effect of antiparasitic therapy on the induction of TF by serum from patients with complicated P. falciparum malaria is dependent on a therapy-mediated loss of activation of NF-κB-like proteins in post-treatment patient serum.

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