scholarly journals Transcriptional Regulation ofFucosyltransferase 1Gene Expression in Colon Cancer Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Fumiko Taniuchi ◽  
Koji Higai ◽  
Tomomi Tanaka ◽  
Yutaro Azuma ◽  
Kojiro Matsumoto
Keyword(s):  
Gene Expression ◽  
Colon Cancer ◽  
Transcriptional Regulation ◽  
Binding Site ◽  
Transcriptional Start Site ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Luciferase Assay ◽  
Start Site ◽  
Lewis Y

Theα1,2-fucosyltransferase I (FUT1) enzyme is important for the biosynthesis of H antigens, Lewis B, and Lewis Y. In this study, we clarified the transcriptional regulation of FUT1 in the DLD-1 colon cancer cell line, which has high expression of Lewis B and Lewis Y antigens, expresses theFUT1gene, and showsα1,2-fucosyltransferase (FUT) activity. 5′-rapid amplification of cDNA ends revealed a FUT1 transcriptional start site −10 nucleotides upstream of the site registered at NM_000148 in the DataBase of Human Transcription Start Sites (DBTSS). Using the dual luciferase assay,FUT1gene expression was shown to be regulated at the region −91 to −81 nt to the transcriptional start site, which contains the Elk-1 binding site. Site-directed mutagenesis of this region revealed the Elk-1 binding site to be essential for FUT1 transcription. Furthermore, transfection of the dominant negative Elk-1 gene, and the chromatin immunoprecipitation (CHIp) assay, supported Elk-1-dependent transcriptional regulation ofFUT1gene expression in DLD-1 cells. These results suggest that a defined region in the 5′-flanking region of FUT1 is critical for FUT1 transcription and that constitutive gene expression ofFUT1is regulated by Elk-1 in DLD-1 cells.

scholarly journals Characterization of PmfR, the Transcriptional Activator of the pAO1-Borne purU-mabO-folD Operon of Arthrobacter nicotinovorans

2005 ◽  
Vol 187 (9) ◽  
pp. 3062-3070 ◽  
Author(s):  
Calin B. Chiribau ◽  
Cristinel Sandu ◽  
Gabor L. Igloi ◽  
Roderich Brandsch
Keyword(s):  
Binding Site ◽  
Transcriptional Start Site ◽  
Transcriptional Activator ◽  
Open Reading Frames ◽  
Start Site ◽  
Gene Encoding ◽  
Chloramphenicol Resistance ◽  
Nuclease Digestion ◽  
Arthrobacter Nicotinovorans

ABSTRACT Nicotine catabolism by Arthrobacter nicotinovorans is linked to the presence of the megaplasmid pAO1. Genes involved in this catabolic pathway are arranged on the plasmid into gene modules according to function. During nicotine degradation γ-N-methylaminobutyrate is formed from the pyrrolidine ring of nicotine. Analysis of the pAO1 open reading frames (ORF) resulted in identification of the gene encoding a demethylating γ-N-methylaminobutyrate oxidase (mabO). This gene was shown to form an operon with purU- and folD-like genes. Only in bacteria grown in the presence of nicotine could transcripts of the purU-mabO-folD operon be detected, demonstrating that this operon constitutes part of the pAO1 nicotine regulon. Its transcriptional start site was determined by primer extension analysis. Transcription of the operon was shown to be controlled by a new transcriptional regulator, PmfR, the product of a gene that is transcribed divergently from the purU, mabO, and folD genes. PmfR was purified, and electromobility shift assays and DNase I-nuclease digestion experiments were used to determine that its DNA binding site is located between −48 and −88 nucleotides upstream of the transcriptional start site of the operon. Disruption of pmfR by homologous recombination with a chloramphenicol resistance cassette demonstrated that PmfR acts in vivo as a transcriptional activator. Mutagenesis of the PmfR target DNA suggested that the sequence GTTT-14 bp-AAAC is the core binding site of the regulator upstream of the −35 promoter region of the purU-mabO-folD operon.

scholarly journals A Putative Binding Site for Sp1 Is Involved in Transcriptional Regulation of CYP17 Gene Expression in Bovine Ovary*

Endocrinology ◽  
1997 ◽  
Vol 138 (5) ◽  
pp. 2011-2020 ◽  
Author(s):  
Raffaella Borroni ◽  
Zheng Liu ◽  
Evan R. Simpson ◽  
Margaret M. Hinshelwood
Keyword(s):  
Gene Expression ◽  
Transcriptional Regulation ◽  
Binding Site ◽  
Cyp17 Gene ◽  
Bovine Ovary ◽  
Putative Binding Site

scholarly journals Extracellular Signal-Regulated Kinase Induces the Megakaryocyte GPIIb/CD41 Gene through MafB/Kreisler

2004 ◽  
Vol 24 (10) ◽  
pp. 4534-4545 ◽  
Author(s):  
Joel R. Sevinsky ◽  
Anne M. Whalen ◽  
Natalie G. Ahn
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Transcriptional Start Site ◽  
Dominant Negative ◽  
Proximal Promoter ◽  
Specific Gene ◽  
Promoter Regulation ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Megakaryocyte Differentiation

ABSTRACT Extracellular signal-regulated kinase (ERK) facilitates cell cycle progression in most mammalian cells, but in certain cell types prolonged signaling through this pathway promotes differentiation and lineage-specific gene expression through mechanisms that are poorly understood. Here, we characterize the transcriptional regulation of platelet GPIIb integrin (CD41) by ERK during megakaryocyte differentiation. ERK-dependent transactivation involves the proximal promoter of GPIIb within 114 bp upstream of the transcriptional start site. GATA, Ets, and Sp1 consensus sequences within this region are each necessary and function combinatorially in ERK-activated transcription. MafB/Kreisler is induced in response to ERK and synergizes with GATA and Ets to enhance transcription from the proximal promoter. The requirement for MafB in promoter regulation is demonstrated by inhibition of transactivation following dominant-negative or antisense suppression of MafB function. Thus, ERK promotes megakaryocyte differentiation by coordinate regulation of nuclear factors that synergize in GPIIb promoter regulation. These results establish a novel role for MafB as a regulator of ERK-induced gene expression.

scholarly journals Protein Kinase A and Mitogen-Activated Protein Kinase Pathways Antagonistically Regulate Fission Yeast fbp1Transcription by Employing Different Modes of Action at Two Upstream Activation Sites

2000 ◽  
Vol 20 (17) ◽  
pp. 6426-6434 ◽  
Author(s):  
Lori A. Neely ◽  
Charles S. Hoffman
Keyword(s):  
Signal Transduction ◽  
Transcriptional Regulation ◽  
Protein Kinase ◽  
Mapk Pathway ◽  
Transcriptional Start Site ◽  
Mitogen Activated Protein Kinase ◽  
Signal Transduction Pathways ◽  
Start Site ◽  
Mitogen Activated Protein ◽  
Kinase A

ABSTRACT A significant challenge to our understanding of eukaryotic transcriptional regulation is to determine how multiple signal transduction pathways converge on a single promoter to regulate transcription in divergent fashions. To study this, we have investigated the transcriptional regulation of theSchizosaccharomyces pombe fbp1 gene that is repressed by a cyclic AMP (cAMP)-dependent protein kinase A (PKA) pathway and is activated by a stress-activated mitogen-activated protein kinase (MAPK) pathway. In this study, we identified and characterized twocis-acting elements in the fbp1 promoter required for activation of fbp1 transcription. Upstream activation site 1 (UAS1), located approximately 900 bp from the transcriptional start site, resembles a cAMP response element (CRE) that is the binding site for the atf1-pcr1 heterodimeric transcriptional activator. Binding of this activator to UAS1 is positively regulated by the MAPK pathway and negatively regulated by PKA. UAS2, located approximately 250 bp from the transcriptional start site, resembles a Saccharomyces cerevisiae stress response element. UAS2 is bound by transcriptional activators and repressors regulated by both the PKA and MAPK pathways, although atf1 itself is not present in these complexes. Transcriptional regulation offbp1 promoter constructs containing only UAS1 or UAS2 confirms that the PKA and MAPK regulation is targeted to both sites. We conclude that the PKA and MAPK signal transduction pathways regulatefbp1 transcription at UAS1 and UAS2, but that the antagonistic interactions between these pathways involve different mechanisms at each site.

Homocysteine-responsive ATF3 gene expression in human vascular endothelial cells: activation of c-Jun NH2-terminal kinase and promoter response element

Blood ◽  
2000 ◽  
Vol 96 (6) ◽  
pp. 2140-2148 ◽  
Author(s):  
Yong Cai ◽  
Chun Zhang ◽  
Tigre Nawa ◽  
Teijiro Aso ◽  
Makiko Tanaka ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Protein Kinase ◽  
Transcriptional Start Site ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Transcriptional Level ◽  
Appreciable Effect ◽  
Responsive Element ◽  
Atf3 Expression

Abstract Activating transcription factor (ATF) 3 is a member of ATF/cyclic adenosine monophosphate (cAMP)–responsive element binding protein (ATF/CREB) family of transcription factors and functions as a stress-inducible transcriptional repressor. To understand the stress-induced gene regulation by homocysteine, we investigated activation of the ATF3 gene in human endothelial cells. Homocysteine caused a rapid induction of ATF3 at the transcriptional level. This induction was preceded by a rapid and sustained activation of c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK), and dominant negative mitogen-activated protein kinase kinase 4 and 7 abolished these effects. The effect of homocysteine appeared to be specific, because cysteine or homocystine had no appreciable effect, but it was mimicked by dithiothreitol and β-mercaptoethanol as well as tunicamycin. The homocysteine effect was not inhibited by an active oxygen scavenger. Deletion analysis of the 5′ flanking sequence of the ATF3 gene promoter revealed that one of the major elements responsible for the induction by homocysteine is an ATF/cAMP responsive element (CRE) located at −92 to −85 relative to the transcriptional start site. Gel shift, immunoprecipitation, and cotransfection assays demonstrated that a complex (or complexes) containing ATF2, c-Jun, and ATF3 increased binding to the ATF/CRE site in the homocysteine-treated cells and activated the ATF3 gene expression, while ATF3 appeared to repress its own promoter. These data together suggested a novel pathway by which homocysteine causes the activation of JNK/SAPK and subsequent ATF3 expression through its reductive stress. Activation of JNK/SAPK and ATF3 expression in response to homocysteine may have a functional role in homocysteinemia-associated endothelial dysfunction.

scholarly journals Full-Length Sequence of Mouse Acupuncture-Induced 1-L (Aig1l) Gene Including Its Transcriptional Start Site

2011 ◽  
Vol 2011 ◽  
pp. 1-7
Author(s):  
Mika Ohta ◽  
Aki Sugano ◽  
Shuji Goto ◽  
Surini Yusoff ◽  
Yushi Hirota ◽  
...  
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Polymerase Chain Reaction ◽  
Tissue Distribution ◽  
Cdna Sequence ◽  
Transcriptional Start Site ◽  
Full Length ◽  
Start Site ◽  
Chain Reaction ◽  
Polymerase Chain

We have been investigating the molecular efficacy of electroacupuncture (EA), which is one type of acupuncture therapy. In our previous molecular biological study of acupuncture, we found an EA-induced gene, named acupuncture-induced 1-L (Aig1l), in mouse skeletal muscle. The aims of this study consisted of identification of the full-length cDNA sequence ofAig1lincluding the transcriptional start site, determination of the tissue distribution ofAig1land analysis of the effect of EA onAig1lgene expression. We determined the complete cDNA sequence including the transcriptional start site via cDNA cloning with the cap site hunting method. We then analyzed the tissue distribution ofAig1lby means of northern blot analysis and real-time quantitative polymerase chain reaction. We used the semiquantitative reverse transcriptase-polymerase chain reaction to examine the effect of EA onAig1lgene expression. Our results showed that the complete cDNA sequence ofAig1lwas 6073 bp long, and the putative protein consisted of 962 amino acids. All seven tissues that we analyzed expressed theAig1lgene. In skeletal muscle, EA induced expression of theAig1lgene, with high expression observed after 3 hours of EA. Our findings thus suggest that theAig1lgene may play a key role in the molecular mechanisms of EA efficacy.

scholarly journals Characterization of CorR, a Transcriptional Activator Which Is Required for Biosynthesis of the Phytotoxin Coronatine

1998 ◽  
Vol 180 (23) ◽  
pp. 6252-6259 ◽  
Author(s):  
Alejandro Peñaloza-Vázquez ◽  
Carol L. Bender
Keyword(s):  
Gene Expression ◽  
Gene Cluster ◽  
Pseudomonas Syringae ◽  
Transcriptional Start Site ◽  
Regulatory Region ◽  
Dnase I ◽  
Upstream Region ◽  
Start Site ◽  
Large Area ◽  
Coronafacic Acid

ABSTRACT Coronatine (COR) is a plasmid-encoded phytotoxin synthesized by several pathovars of phytopathogenic Pseudomonas syringae. The COR biosynthetic gene cluster in P. syringae pv. glycinea PG4180 is encoded by a 32-kb region which contains both the structural and regulatory genes needed for COR synthesis. The regulatory region contains three genes: corP,corS, and corR. corS is thought to function as a histidine protein kinase, whereas corP andcorR show relatedness to response regulators of the two-component regulatory paradigm. In the present study, we investigated whether CorR is a positive activator of COR gene expression. We also studied whether CorR specifically binds the DNA region located upstream of cfl, a gene located at the 5′ end of the gene cluster encoding coronafacic acid, the polyketide portion of COR. Complementation analysis with a corRmutant, PG4180.P2, and transcriptional fusions to a promoterless glucuronidase gene (uidA) indicated that CorR functions as a positive regulator of COR gene expression. Deletion analysis of the 5′ end of the cfl upstream region was used to define the minimal region required for COR gene expression. A 360-bp DNA fragment located over 500 bp upstream from the cfl transcriptional start site was used in DNase I protection assays to define the specific bases bound by CorR. An area extending from −704 to −650 with respect to the cfl transcriptional start site was protected by DNase I footprinting, indicating a rather large area of protection. This area was also conserved in the promoter region forcmaA, which encodes a transcript containing genes for coronamic acid synthesis, another intermediate in the COR biosynthetic pathway. The results obtained in the current study suggest that both the coronafacic acid and the coronamic acid structural genes are controlled by CorR, a positive activator of COR gene expression.

scholarly journals Transcriptional Repression Mediated by LysR-Type Regulator CatR Bound at Multiple Binding Sites

1998 ◽  
Vol 180 (9) ◽  
pp. 2367-2372 ◽  
Author(s):  
Sudha A. Chugani ◽  
Matthew R. Parsek ◽  
A. M. Chakrabarty
Keyword(s):  
Binding Site ◽  
Structural Gene ◽  
Binding Sites ◽  
Transcriptional Start Site ◽  
Dnase I ◽  
Site Directed Mutagenesis ◽  
Dnase I Footprinting ◽  
Gel Shift

ABSTRACT The catBCA operon of Pseudomonas putidaencodes enzymes involved in the catabolism of benzoate. Transcription of this operon requires the LysR-type transcriptional regulator CatR and an inducer molecule, cis,cis-muconate. Previous gel shift assays and DNase I footprinting have demonstrated that CatR occupies two adjacent sites proximal to thecatBCA promoter in the presence of the inducer. We report the presence of an additional binding site for CatR downstream of thecatBCA promoter within the catB structural gene. This site, called the internal binding site (IBS), extends from +162 to +193 with respect to the catB transcriptional start site and lies within the catB open reading frame. Gel shift analysis and DNase I footprinting determined that CatR binds to this site with low affinity. CatR binds cooperatively with higher affinity to the IBS in the presence of the two upstream binding sites. Parallel in vivo and in vitro studies were conducted to determine the role of the internal binding site. We measured β-galactosidase activity ofcatB-lacZ transcriptional fusions in vivo. Our results suggest a probable cis-acting repressor function for the internal binding site. Site-directed mutagenesis of the IBS verified this finding. The location of the IBS within the catBstructural gene, the cooperativity observed in footprinting studies, and phasing studies suggest that the IBS likely participates in the interaction of CatR with the upstream binding sites by looping out the intervening DNA.

scholarly journals Identification and transcriptional regulation of the mir-218-1 alternative promoter

2020 ◽  
Author(s):  
Brenna A. Rheinheimer ◽  
Lukas Vrba ◽  
Bernard W Futscher ◽  
Ronald L Heimark
Keyword(s):  
Transcriptional Regulation ◽  
Pancreatic Ductal Adenocarcinoma ◽  
Chromatin Immunoprecipitation ◽  
Transcriptional Start Site ◽  
Regulatory Elements ◽  
Alternative Promoter ◽  
Ductal Adenocarcinoma ◽  
Start Site ◽  
Public Datasets ◽  
Intron 4

AbstractBackgroundmiRNAs are small, endogenous non-coding RNAs approximately 22 nucleotides in length that account for approximately 1% of the genome and play key regulatory roles in multiple signaling pathways. mir-218-1 is an intronic miRNA located within intron 15 of the SLIT2 gene. Public datasets showed enrichment of H3K4me3 within intron 4 of the SLIT2 gene. Therefore, we sought to determine the genomic location and transcriptional regulatory elements of the mir-218-1 candidate alternative promoter in pancreatic ductal adenocarcinoma.MethodsExpression of mir-218 was evaluated in a panel of pancreatic ductal adenocarcinoma cell lines. The mir-218-1 candidate alternative promoter was characterized by chromatin immunoprecipitation, Sequenom, and luciferase assays. Transcriptional regulation of the mir-218-1 candidate alternative promoter was assessed using chromatin immunoprecipitation and an inhibitor to NF-kB.ResultsWe found that expression of mir-218-1 does not correlate with SLIT2 expression and that mir-218-1 has a novel transcriptional start site separate from the SLIT2 promoter. This novel transcriptional start site showed transcriptional activity and was regulated by NF-kB.Conclusionsmir-218-1 is transcribed from an independent and novel transcriptional start site located within intron 4 of the SLIT2 gene in pancreatic ductal adenocarcinoma. Additionally, mir-218-1 expression is regulated by Nf-kB at this alternative transcriptional start site in pancreatic cancer.

scholarly journals Identification of σE-Dependent Promoter Upstream of clpB from the Pathogenic Spirochaete Leptospira interrogans by Applying an E. coli Two-Plasmid System

2019 ◽  
Vol 20 (24) ◽  
pp. 6325
Author(s):  
Sabina Kędzierska-Mieszkowska ◽  
Katarzyna Potrykus ◽  
Zbigniew Arent ◽  
Joanna Krajewska
Keyword(s):  
Gene Expression ◽  
Gene Expression Regulation ◽  
Transcriptional Start Site ◽  
Leptospira Interrogans ◽  
Start Site ◽  
Limited Information ◽  
E Coli ◽  
Oxidative Stresses ◽  
Genetic Mechanisms ◽  
Site Identification

There is limited information on gene expression in the pathogenic spirochaete Leptospira interrogans and genetic mechanisms controlling its virulence. Transcription is the first step in gene expression that is often determined by environmental effects, including infection-induced stresses. Alterations in the environment result in significant changes in the transcription of many genes, allowing effective adaptation of Leptospira to mammalian hosts. Thus, promoter and transcriptional start site identification are crucial for determining gene expression regulation and for the understanding of genetic regulatory mechanisms existing in Leptospira. Here, we characterized the promoter region of the L. interrogans clpB gene (clpBLi) encoding an AAA+ molecular chaperone ClpB essential for the survival of this spirochaete under thermal and oxidative stresses, and also during infection of the host. Primer extension analysis demonstrated that transcription of clpB in L. interrogans initiates at a cytidine located 41 bp upstream of the ATG initiation codon, and, to a lesser extent, at an adenine located 2 bp downstream of the identified site. Transcription of both transcripts was heat-inducible. Determination of clpBLi transcription start site, combined with promoter transcriptional activity assays using a modified two-plasmid system in E. coli, revealed that clpBLi transcription is controlled by the ECF σE factor. Of the ten L. interrogans ECF σ factors, the factor encoded by LIC_12757 (LA0876) is most likely to be the key regulator of clpB gene expression in Leptospira cells, especially under thermal stress. Furthermore, clpB expression may be mediated by ppGpp in Leptospira.

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