scholarly journals TLR4 promoter rs1927914 variant contributes to the susceptibility of esophageal squamous cell carcinoma in the Chinese population

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e10754
Author(s):  
Jiaying Li ◽  
Hongjiao Wu ◽  
Hui Gao ◽  
Ruihuan Kou ◽  
Yuning Xie ◽  
...  
Keyword(s):  
Esophageal Cancer ◽  
Statistical Significance ◽  
Nuclear Extract ◽  
Taqman Probe ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Link Type ◽  
Pcr Rflp ◽  
Dna Protein Interaction ◽  
Mobility Shift Assay

Background Toll-like receptor 4 (TLR4), as a key regulator of both innate and acquired immunity, has been linked with the development of various cancers, including esophageal cancer. This study aims to analyze the association of potential functional genetic polymorphisms in TLR4 with the risk of esophageal cancer. Methods This case-control study involved in 480 ESCC patients and 480 health controls. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to genotype TLR4 rs1927914 polymorphism. Taqman probe method was used to determine the genotypes of TLR4 rs11536891 and rs7873784 variants. The relationship between TLR4 genetic variation and ESCC risk was analyzed by Logistic regression model by calculating the odds ratio (OR) and 95% confidence interval (95% CI). Results Compared with TLR4 rs1927914 AA genotype carriers, GG carriers had a lower ESCC risk (OR = 0.59, 95% CI [0.38–0.93], P = 0.023). Stratification analysis by age showed that TLR4 rs1927914 GG could affect the risk of ESCC in elderly people (OR = 0.59, 95% CI [0.36–0.97]). Smoking stratification analysis indicated that rs1927914 GG carriers were related to ESCC susceptibility among non-smokers (OR = 0.36, 95% CI [0.18–0.73]). Dual luciferase reporter assay suggested that rs1927914 G-containing TLR4 promoter displayed a 1.76-fold higher luciferase activity than rs1927914 A-containing counterpart in KYSE30 cells. Electrophoretic mobility shift assay (EMSA) showed the KYSE30 cell nuclear extract was able to bind the probe with rs1927914 G allele and this DNA-protein interaction could be eliminated by competition assays with unlabeled rs1927914 G probe, which indicating that the binding is sequence-specific. Our results also showed that TLR4 rs7873784 (G>C) and rs11536891 (T>C) conformed to complete genetic linkage. The genotype distributions of TLR4 rs11536891 variant among ESCC patients and normal controls have no statistical significance. Conclusion The TLR4 rs1927914 variant contributes to the ESCC risk by effecting the promoter activity.

DNA-binding factors of B lymphoid cells are susceptible to limited proteolytic cleavage during nuclear extract preparation

1988 ◽  
Vol 8 (4) ◽  
pp. 1812-1815
Author(s):  
E L Mather
Keyword(s):  
Dna Binding ◽  
Nuclear Extract ◽  
Lymphoid Cells ◽  
Chain Gene ◽  
Immunoglobulin Heavy Chain Gene ◽  
Mobility Shift ◽  
Lymphoid Lines ◽  
Dna Binding Factors ◽  
Mobility Shift Assay ◽  
B Lymphoid

DNA-binding proteins that interact with the 3' end of the mouse mu immunoglobulin heavy chain gene were identified by the electrophoretic mobility shift assay. Complexes of distinctly different mobilities were formed by extracts prepared from B lymphoid lines representing different stages of maturation. The apparent stage-specific differences are shown to be due to proteolytic events that occurred during extract preparation.

A requirement for thioredoxin in redox-sensitive modulation of T-cadherin expression in endothelial cells

2008 ◽  
Vol 416 (2) ◽  
pp. 271-280 ◽  
Author(s):  
Manjunath B. Joshi ◽  
Danila Ivanov ◽  
Maria Philippova ◽  
Emmanouil Kyriakakis ◽  
Paul Erne ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Endothelial Cells ◽  
Nuclear Extract ◽  
Mobility Shift ◽  
Protein Levels ◽  
Nuclear Extracts ◽  
Nucleoprotein Complex ◽  
Thioredoxin 1 ◽  
Mobility Shift Assay ◽  
Reporter Gene Analysis

T-cad (T-cadherin), a glycosylphosphatidylinositol-anchored cadherin superfamily member, is expressed widely in the brain and cardiovascular system, and absent, decreased, or even increased, in cancers. Mechanisms controlling T-cad expression are poorly understood. The present study investigated transcriptional regulation of T-cad in ECs (endothelial cells). Conditions of oxidative stress (serum-deprivation or presence of H2O2) elevate T-cad mRNA and protein levels in ECs. Reporter gene analysis, using serially deleted T-cad promoter stretches ranging from −99 to −2304 bp, located the minimal promoter region of T-cad within −285 bp from the translation start site. Reporter activity in ECs transfected with the −285 bp construct increased under conditions of oxidative stress, and this was normalized by antioxidant N-acetylcysteine. An electrophoretic-mobility-shift assay revealed a specific nucleoprotein complex unique to −156 to −203 bp, which increased when nuclear extracts from oxidatively stressed ECs were used, suggesting the presence of redox-sensitive binding element(s). MS analysis of the nucleoprotein complex unique to −156 to −203 bp after streptavidin–agarose pull-down detected the presence of the redox-active protein thioredoxin. The presence of thioredoxin-1 in a nuclear extract from oxidatively stressed ECs was demonstrated after immunoprecipitation and immunoblotting. Transfection of ECs with thioredoxin-1 small interfering RNA abrogated oxidative-stress-induced up-regulation of T-cad transcripts and protein. We conclude that thioredoxin-1 is an important determinant of redox-sensitive transcriptional up-regulation of T-cad in ECs.

scholarly journals Downregulation of PU.1 Leads to Decreased Expression of Dectin-1 in Alveolar Macrophages during Pneumocystis Pneumonia

2010 ◽  
Vol 78 (3) ◽  
pp. 1058-1065 ◽  
Author(s):  
Chen Zhang ◽  
Shao-Hung Wang ◽  
Chung-Ping Liao ◽  
Shoujin Shao ◽  
Mark E. Lasbury ◽  
...  
Keyword(s):  
Alveolar Macrophages ◽  
Mrna Level ◽  
Gene Promoter ◽  
Pneumocystis Pneumonia ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Mobility Shift Assay

ABSTRACT Dectin-1 is an important macrophage phagocytic receptor recognizing fungal β-glucans. In this study, the mRNA levels of the Dectin-1 gene were found to be decreased by 61% in alveolar macrophages (AMs) from Pneumocystis-infected mice. The expression of Dectin-1 protein on the surface of these cells was also significantly decreased. By fluorescence in situ hybridization, mRNA expression levels of the transcription factor PU.1 were also found to be significantly reduced in AMs from Pneumocystis-infected mice. Electrophoretic mobility shift assay showed that PU.1 protein bound Dectin-1 gene promoter. With a luciferase reporter gene driven by the Dectin-1 gene promoter, the expression of the PU.1 gene in NIH 3T3 cells was found to enhance the luciferase activity in a dose-dependent manner. PU.1 expression knockdown by small interfering RNA (siRNA) caused a 63% decrease in Dectin-1 mRNA level and 40% decrease in protein level in AMs. Results of this study indicate that downregulation of PU.1 during Pneumocystis pneumonia leads to decreased expression of Dectin-1 in AMs.

scholarly journals Detection of damage-recognition proteins from human lymphocytes.

2000 ◽  
Vol 47 (2) ◽  
pp. 443-450 ◽  
Author(s):  
J Lanuszewska ◽  
A Cudak ◽  
J Rzeszowska-Wolny ◽  
P Widłak
Keyword(s):  
Electrophoretic Mobility ◽  
Human Lymphocytes ◽  
Nuclear Extract ◽  
Mobility Shift ◽  
Damage Recognition ◽  
Free Dna ◽  
Healthy Donors ◽  
Mobility Shift Assay ◽  
Dna Ends ◽  
Damaged Dna

Proteins recognizing and binding to damaged DNA (DDB-proteins) were analyzed in human lymphocytes obtained from healthy donors. Using an electrophoretic mobility shift assay several complexes between nuclear extract proteins and damaged DNA were detected: a complex specific for DNA damaged by N-acetoxy-N-acetylaminofluorene, another complex specific for UV-irradiated DNA, and two complexes specific for DNA damaged by cis-dichlorodiammine platinum. All the detected complexes differed in electrophoretic mobility and possibly contained different proteins. Complexes specific for free DNA ends were also detected in protein extracts from lymphocytes.

scholarly journals GATA-1 binding to the alphaV promoter negatively regulates expression of the integrin alphaV subunit in human leukemic K562 cells.

2002 ◽  
Vol 49 (1) ◽  
pp. 19-28 ◽  
Author(s):  
Małgorzata Czyz ◽  
Marta Stasiak ◽  
Joanna Boncela ◽  
Czesław S Cierniewsk
Keyword(s):  
Vascular Endothelial Cells ◽  
Gene Promoter ◽  
K562 Cells ◽  
Nuclear Extract ◽  
Binding Activity ◽  
Vascular Endothelial ◽  
Mobility Shift ◽  
Dnase I Footprinting ◽  
Mobility Shift Assay ◽  
Lower Expression

Recently we observed that the transcription factors Sp1 and Sp3 bind to the CTCCTCCTC sequence located between positions -194 and -172 of the alphaV promoter region and are directly involved in the regulation of transcriptional activity of the alphaV gene in human umbilical vascular endothelial cells (HUVECs) (Czyz & Cierniewski, 1999, Eur. J. Biochem. 265, 638). In this report we provide evidence that the GATA-1 factor regulates alphaV expression during differentiation of pluripotent K562 cells induced either by phorbol 12-myristate 13-acetate (PMA) or butyric acid (BA) through interaction with the GATA element in the alphaV gene promoter. DNase I footprinting analysis revealed that region -413 to -408, covering the GATA binding site, was protected by nuclear extract from K562 cells. There was no protection of this region by HUVEC nuclear extract. Electrophoretic mobility shift assay (EMSA) analysis of nuclear extract of K562 cells treated with BA revealed an increase in GATA binding activity, which was associated with reduced alphaV mRNA and alphaV protein on the cell surface. Stimulation of K562 cells with PMA resulted in opposite effects: lower expression of GATA-1 was associated with increased levels of alphaV. We conclude that the GATA-1 transcription factor specifically binds to the GATA element in the alphaV gene promoter and negatively regulates alphaV gene expression.

scholarly journals CCAAT Displacement Protein (CDP/cut) Recognizes a Silencer Element Within the Lactoferrin Gene Promoter

Blood ◽  
1997 ◽  
Vol 90 (7) ◽  
pp. 2784-2795 ◽  
Author(s):  
Arati Khanna-Gupta ◽  
Theresa Zibello ◽  
Sarah Kolla ◽  
Ellis J. Neufeld ◽  
Nancy Berliner
Keyword(s):  
Luciferase Activity ◽  
Gene Promoter ◽  
Luciferase Reporter ◽  
Transcriptional Level ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Ccaat Displacement Protein ◽  
Lactoferrin Gene ◽  
Mobility Shift Assay ◽  
Silencer Element

Abstract Expression of neutrophil secondary granule protein (SGP) genes is coordinately regulated at the transcriptional level, and is disrupted in specific granule deficiency and leukemia. We analyzed the regulation of SGP gene expression by luciferase reporter gene assays using the lactoferrin (LF) promoter. Reporter plasmids were transiently transfected into non–LF-expressing hematopoietic cell lines. Luciferase activity was detected from reporter plasmids containing basepair (bp) −387 to bp −726 of the LF promoter, but not in a −916-bp plasmid. Transfection of a −916-bp plasmid into a LF-expressing cell line resulted in abrogation of the silencing effect. Sequence analysis of this region revealed three eight-bp repetitive elements, the deletion of which restored wild-type levels of luciferase activity to the −916-bp reporter plasmid. Electrophoretic mobility shift assay and UV cross-linking analysis identified a protein of approximately 180 kD that binds to this region in non–LF-expressing cells but not in LF-expressing cells. This protein was identified to be the CCAAT displacement protein (CDP/cut). CDP/cut has been shown to downregulate expression of gp91-phox, a gene expressed relatively early in the myeloid lineage. Our observations suggest that the binding of CDP/cut to the LF silencer element serves to suppress basal promoter activity of the LF gene in non–LF-expressing cells. Furthermore, overexpression of CDP/cut in cultured myeloid stem cells blocks LF expression upon granulocyte colony-stimulating factor–induced neutrophil maturation without blocking phenotypic maturation. This block in LF expression may be due, in part, to the persistence of CDP/cut binding to the LF silencer element.

scholarly journals Heterogeneous nuclear ribonucleoprotein K represses transcription from a cytosine/thymidine-rich element in the osteocalcin promoter

2005 ◽  
Vol 385 (2) ◽  
pp. 613-623 ◽  
Author(s):  
Joseph P. STAINS ◽  
Fernando LECANDA ◽  
Dwight A. TOWLER ◽  
Roberto CIVITELLI
Keyword(s):  
Gene Transcription ◽  
Nuclear Extract ◽  
Binding Activity ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Hnrnp K ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Osteocalcin Gene ◽  
Nuclear Ribonucleoprotein

HnRNP K (heterogeneous nuclear ribonucleoprotein K) was biochemically purified from a screen of proteins co-purifying with binding activity to the osteocalcin promoter. We identify hnRNP K as a novel repressor of osteocalcin gene transcription. Overexpression of hnRNP K lowers the expression of osteocalcin mRNA by 5-fold. Furthermore, luciferase reporter assays demonstrate that overexpression of hnRNP K represses osteocalcin transcription from a CT (cytosine/thymidine)-rich element in the proximal promoter. Electrophoretic mobility-shift analysis reveals that recombinant hnRNP K binds to the CT-rich element, but binds ss (single-stranded), rather than ds (double-stranded) oligonucleotide probes. Accordingly, hnRNP K antibody can supershift a binding activity present in nuclear extracts using ss sense, but not antisense or ds oligonucleotides corresponding to the CT-rich −95 to −47 osteocalcin promoter. Importantly, addition of recombinant hnRNP K to ROS 17/2.8 nuclear extract disrupts formation of a DNA–protein complex on ds CT element oligonucleotides. This action is mutually exclusive with hnRNP K's ability to bind ss DNA. These results demonstrate that hnRNPK, although co-purified with a dsDNA-binding activity, does not itself bind dsDNA. Rather, hnRNP K represses osteocalcin gene transcription by inhibiting the formation of a transcriptional complex on the CT element of the osteocalcin promoter.

scholarly journals A homozygous point mutation in the GH1 promoter (c.-223C>T) leads to reduced GH1 expression in siblings with isolated GH deficiency (IGHD)

2016 ◽  
Vol 175 (2) ◽  
pp. K7-K15 ◽  
Author(s):  
João L O Madeira ◽  
Alexander A L Jorge ◽  
Regina M Martin ◽  
Luciana R Montenegro ◽  
Marcela M Franca ◽  
...  
Keyword(s):  
Point Mutation ◽  
Nuclear Extract ◽  
University Hospital ◽  
Proximal Promoter ◽  
Hormone Deficiency ◽  
Luciferase Reporter ◽  
Pituitary Cells ◽  
Nuclear Extracts ◽  
Reporter Assays ◽  
Dna Protein Interaction

Context Mutations in the GH1 promoter are a rare cause of isolated growth hormone deficiency (IGHD). Objective To identify the molecular aetiology of a family with IGHD. Design DNA sequencing, electromobility shift (EMSA) and luciferase reporter assays. Setting University Hospital. Patients Three siblings (2M) born to consanguineous parents presented with IGHD with normal pituitary on MRI. Methods The GH1 proximal promoter, locus control region, five exons and four introns as well as GHRHR gene were sequenced in genomic DNA by Sanger method. DNA–protein interaction was evaluated by EMSA in nuclear extracts of GH3 pituitary cells. Dual-luciferase reporter assays were performed in cells transiently transfected with plasmids containing four different combinations of GH1 allelic variants (AV). Results The patients harboured two homozygous variants (c.-185T>C and c.-223C>T) in the GH1 promoter within a highly conserved region and predicted binding sites for POU1F1/SP1 and SP1 respectively. The parents and brother were carriers and these variants were absent in 100 controls. EMSA demonstrated absent binding of GH3 nuclear extract to the c.-223C>T variant and normal binding of both POU1F1 protein and GH3 nuclear extract to the c.-185T>C variant. In contrast to GH1 promoter with AV only at c.-185, the GH1 promoter containing the AV only at c.-223 and at both positions drove significantly less expression of luciferase compared with the promoter containing either positions wild type in luciferase reporter assays. Conclusion To our knowledge, c.-223C>T is the first homozygous point mutation in the GH1 promoter that leads to short stature due to IGHD.

scholarly journals DNA-binding factors of B lymphoid cells are susceptible to limited proteolytic cleavage during nuclear extract preparation.

1988 ◽  
Vol 8 (4) ◽  
pp. 1812-1815 ◽  
Author(s):  
E L Mather
Keyword(s):  
Dna Binding ◽  
Nuclear Extract ◽  
Lymphoid Cells ◽  
Chain Gene ◽  
Immunoglobulin Heavy Chain Gene ◽  
Mobility Shift ◽  
Lymphoid Lines ◽  
Dna Binding Factors ◽  
Mobility Shift Assay ◽  
B Lymphoid

DNA-binding proteins that interact with the 3' end of the mouse mu immunoglobulin heavy chain gene were identified by the electrophoretic mobility shift assay. Complexes of distinctly different mobilities were formed by extracts prepared from B lymphoid lines representing different stages of maturation. The apparent stage-specific differences are shown to be due to proteolytic events that occurred during extract preparation.

scholarly journals Centromere protein B assembles human centromeric alpha-satellite DNA at the 17-bp sequence, CENP-B box.

1992 ◽  
Vol 116 (3) ◽  
pp. 585-596 ◽  
Author(s):  
Y Muro ◽  
H Masumoto ◽  
K Yoda ◽  
N Nozaki ◽  
M Ohashi ◽  
...  
Keyword(s):  
Nuclear Extract ◽  
Affinity Column ◽  
Alpha Satellite ◽  
Mobility Shift ◽  
Alpha Satellite Dna ◽  
Centromere Protein ◽  
Alphoid Dna ◽  
Dna Mobility ◽  
Mobility Shift Assay ◽  
Protein B

We purified 15,000-fold from HeLa cell nuclear extract the centromere antigen that reacts specifically with the 17-bp sequence, designated previously as CENP-B box, in human centromeric alpha-satellite (alphoid) DNA by a two-step procedure including an oligonucleotide affinity column. The purified protein was identified as the centromere protein B (CENP-B) by its mobility on SDS-PAGE (80 kD), and reactivities to a monoclonal antibody raised to CENP-B (bacterial fusion protein) and to anticentromere sera from patients with autoimmune diseases. Direct binding by CENP-B of the CENP-B box sequence in the alphoid DNA has been proved using the purified CENP-B by DNA mobility-shift assay, Southwestern blotting, and DNase I protection analysis. The binding constant of the antigen to the CENP-B box sequence is 6 x 10(8) M-1. DNA mobility-shift assays indicated that the major complex formed between the CENP-B and the DNA contains two DNA molecules, suggesting the importance of the CENP-B/CENP-B box interaction in organization of higher ordered chromatin structures in the centromere and/or kinetochore. Location of DNA binding and dimerization domains in CENP-B was discussed based on the DNA mobility-shift assays performed with a protein fraction containing intact and partial cleavage products of CENP-B.

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