Genomic organization, chromosomal mapping and promoter analysis of the mouse selenocysteine tRNA gene transcription-activating factor (mStaf) gene

mStaf is a zinc-finger protein that activates the transcription of the mouse selenocysteine tRNA gene. The mStaf gene is approx. 35 kb long and split into 16 exons. All exon-intron junction sequences conform to the GT/AG rule. The transcription start site is located 83 bp upstream of the initiation codon. Chromosomal mapping localized the gene to mouse chromosome 7, region E3-F1. Sequence analysis of the proximal promoter region revealed several potential regulatory elements; these include the recognition elements of Sp1, Nkx, CP2, E2A, SIF (SIS-inducible factor), TFII-I and cAMP-responsive element (CRE), but no TATA sequences. Transfection experiments demonstrated that the 5ʹ-flanking region (-1894 to +37) of the mStaf gene drives transcription in mouse NMuMG cells and that a construct containing a fragment from -387 to +37 showed the highest transcriptional activity. Deletion and mutation experiments suggested that four Sp1 sites played an important role for the basal promoter activity. Furthermore, electrophoretic mobility-shift assays demonstrated that Sp3 but not other Sp (specificity protein) family members binds to three of the Sp1 sites. Our present study suggests that Sp3 is involved in the basal transcriptional activation of the mStaf gene.

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SOX17 directly activates Zfp202 transcription during in vitro endoderm differentiation

Physiological Genomics ◽

10.1152/physiolgenomics.90236.2008 ◽

2008 ◽

Vol 34 (3) ◽

pp. 277-284 ◽

Cited By ~ 5

Author(s):

Ethan S. Patterson ◽

Russell C. Addis ◽

Michael J. Shamblott ◽

John D. Gearhart

Keyword(s):

Transcriptional Activation ◽

Zinc Finger Protein ◽

High Mobility ◽

Mobility Shift ◽

F9 Cells ◽

Endoderm Differentiation ◽

F9 Embryonal Carcinoma Cells ◽

Multiple Species ◽

Electrophoretic Mobility Shift Assays

SOX17 is a SRY-related high-mobility group (HMG) box transcription factor that is necessary for endoderm formation in multiple species. Despite its essential function during endoderm formation and differentiation, few direct targets of SOX17 are known. To identify targets of SOX17, we isolated SOX17 binding sites with a chromatin immunoprecipitation (ChIP)-cloning screen. SOX17-ChIP identified zinc finger protein 202 ( Zfp202) as a direct target of SOX17 during endoderm differentiation of F9 embryonal carcinoma cells. A sequence in the first intron of Zfp202 activated transcription in differentiated F9 cells, and overexpression of Sox17 increased the transcriptional activity of this sequence. SOX17 binds to a site within this sequence in electrophoretic mobility shift assays, and mutation of this site decreases the transcriptional activation. Zfp202 is induced concomitantly with Sox17 during endoderm differentiation of F9 cells. We also show that ZFP202 represses Hnf4a, which has been reported for the human ortholog ZNF202. Identifying targets of SOX17 will help to elucidate the molecular basis of endoderm differentiation and may provide a better understanding of the role of endoderm in patterning the other germ layers.

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An IRP-like protein from Plasmodium falciparum binds to a mammalian iron-responsive element

Blood ◽

10.1182/blood.v98.8.2555 ◽

2001 ◽

Vol 98 (8) ◽

pp. 2555-2562 ◽

Cited By ~ 26

Author(s):

Mark Loyevsky ◽

Timothy LaVaute ◽

Charles R. Allerson ◽

Robert Stearman ◽

Olakunle O. Kassim ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Regulatory Protein ◽

Protein S ◽

Fold Increase ◽

Binding Activity ◽

Mobility Shift ◽

Iron Responsive Element ◽

Element Binding Protein ◽

Responsive Element ◽

Electrophoretic Mobility Shift Assays

Abstract This study cloned and sequenced the complementary DNA (cDNA) encoding of a putative malarial iron responsive element-binding protein (PfIRPa) and confirmed its identity to the previously identified iron-regulatory protein (IRP)–like cDNA from Plasmodium falciparum. Sequence alignment showed that the plasmodial sequence has 47% identity with human IRP1. Hemoglobin-free lysates obtained from erythrocyte-stage P falciparum contain a protein that binds a consensus mammalian iron-responsive element (IRE), indicating that a protein(s) with iron-regulatory activity was present in the lysates. IRE-binding activity was found to be iron regulated in the electrophoretic mobility shift assays. Western blot analysis showed a 2-fold increase in the level of PfIRPa in the desferrioxamine-treated cultures versus control or iron-supplemented cells. Malarial IRP was detected by anti-PfIRPa antibody in the IRE-protein complex fromP falciparum lysates. Immunofluorescence studies confirmed the presence of PfIRPa in the infected red blood cells. These findings demonstrate that erythrocyte P falciparum contains an iron-regulated IRP that binds a mammalian consensus IRE sequence, raising the possibility that the malaria parasite expresses transcripts that contain IREs and are iron-dependently regulated.

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Characterization of the promoter elements required for hepatic and intestinal transcription of the human apoB gene: definition of the DNA-binding site of a tissue-specific transcriptional factor.

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.2653 ◽

1990 ◽

Vol 10 (6) ◽

pp. 2653-2659 ◽

Cited By ~ 33

Author(s):

D Kardassis ◽

M Hadzopoulou-Cladaras ◽

D P Ramji ◽

R Cortese ◽

V I Zannis ◽

...

Keyword(s):

Binding Site ◽

Regulatory Elements ◽

Apob Gene ◽

Mobility Shift ◽

Promoter Elements ◽

Nuclear Extracts ◽

Dna Binding Site ◽

Gene Definition ◽

Definition Of ◽

Electrophoretic Mobility Shift Assays

The promoter elements important for intestinal and hepatic transcription of the human apoB gene have been localized downstream of nucleotide -150. Footprinting analysis using hepatic nuclear extracts identified four protected regions, -124 to -100, -97 to -93, -86 to -33, and +33 to +52. Gel electrophoretic mobility shift assays showed that multiple factors interact with the apoB sequence -86 to -33, while the region -88 to -61 binds a single nuclear factor. Methylation interference analysis and nucleotide substitution mutagenesis identified the binding site of the factor between residues -78 and -68. Binding competition experiments indicate that this factor recognizes the regulatory elements of other liver-specific genes.

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Promoter analysis of the DHCR24 (3β-hydroxysterol Δ24-reductase) gene: characterization of SREBP (sterol-regulatoryelement-binding protein)-mediated activation

Bioscience Reports ◽

10.1042/bsr20120095 ◽

2012 ◽

Vol 33 (1) ◽

Cited By ~ 6

Author(s):

Lidia A. Daimiel ◽

María E. Fernández-Suárez ◽

Sara Rodríguez-Acebes ◽

Lorena Crespo ◽

Miguel A. Lasunción ◽

...

Keyword(s):

Cellular Response ◽

Binding Protein ◽

Regulatory Element ◽

Cholesterol Biosynthesis ◽

Regulatory Elements ◽

Proximal Promoter ◽

Luciferase Reporter ◽

Mobility Shift ◽

Gene Characterization ◽

Cis Acting

DHCR24 (3β-hydroxysterol Δ24-reductase) catalyses the reduction of the C-24 double bond of sterol intermediates during cholesterol biosynthesis. DHCR24 has also been involved in cell growth, senescence and cellular response to oncogenic and oxidative stress. Despite its important roles, little is known about the transcriptional mechanisms controlling DHCR24 gene expression. We analysed the proximal promoter region and the cholesterol-mediated regulation of DHCR24. A putative SRE (sterol-regulatory element) at −98/−90 bp of the transcription start site was identified. Other putative regulatory elements commonly found in SREBP (SRE-binding protein)-targeted genes were also identified. Sterol responsiveness was analysed by luciferase reporter assays of approximately 1 kb 5′-flanking region of the human DHCR24 gene in HepG2 and SK-N-MC cells. EMSAs (electrophoretic mobility-shift assays) and ChIP (chromatin immunoprecipitation) assays demonstrated cholesterol-dependent recruitment and binding of SREBPs to the putative SRE. Given the presence of several CACCC-boxes in the DHCR24 proximal promoter, we assessed the role of KLF5 (Krüppel-like factor 5) in androgen-regulated DHCR24 expression. DHT (dihydrotestosterone) increased DHCR24 expression synergistically with lovastatin. However, DHT was unable to activate the DHCR24 proximal promoter, whereas KLF5 did, indicating that this mechanism is not involved in the androgen-induced stimulation of DHCR24 expression. The results of the present study allow the elucidation of the mechanism of regulation of the DHCR24 gene by cholesterol availability and identification of other putative cis-acting elements which may be relevant for the regulation of DHCR24 expression.

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Constitutive activation of LIL-Stat in adult T-cell leukemia cells

Blood ◽

10.1182/blood.v95.8.2715 ◽

2000 ◽

Vol 95 (8) ◽

pp. 2715-2718 ◽

Cited By ~ 5

Author(s):

Junichi Tsukada ◽

Yoko Toda ◽

Masahiro Misago ◽

Yoshiya Tanaka ◽

Philip E. Auron ◽

...

Keyword(s):

T Cell ◽

Dna Binding ◽

Leukemic Cells ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Mobility Shift ◽

Adult T Cell Leukemia ◽

Nuclear Extracts ◽

Responsive Element ◽

Electrophoretic Mobility Shift Assays

Abstract The activation status of a recently identified STAT (signal transducers and activators of transcription) factor, LIL-Stat (lipopolysaccharide [LPS]/IL-1–inducible Stat) in adult T-cell leukemia (ATL) cells was investigated by electrophoretic mobility shift assays using nuclear extracts of leukemic cells from 7 patients with ATL and a GAS (gamma interferon activation site)-like element termed LILRE (LPS/IL-1–responsive element), which is found in the human prointerleukin 1β (IL1B) gene. Spontaneous DNA binding of LIL-Stat was observed in all ATL cells examined. However, in normal human peripheral lymphocytes, DNA binding of LIL-Stat was detected only after stimulation with IL-1. These results demonstrated that LIL-Stat is constitutively activated in ATL cells. Furthermore, our transient transfection studies using LILRE chloramphenicol acetyltransferase (CAT) reporters argue that LIL-Stat in ATL cells functions as a transcriptional activator through binding to the LILRE in theIL1B gene.

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A natural sequence consisting of overlapping glucocorticoid-responsive elements mediates glucocorticoid, but not androgen, regulation of gene expression

Biochemical Journal ◽

10.1042/bj3500123 ◽

2000 ◽

Vol 350 (1) ◽

pp. 123-129 ◽

Cited By ~ 10

Author(s):

Charbel MASSAAD ◽

Michèle GARLATTI ◽

Elizabeth M. WILSON ◽

Françoise CADEPOND ◽

Robert BAROUKI

Keyword(s):

Gene Expression ◽

Thymidine Kinase ◽

Transcriptional Activation ◽

Regulation Of Gene Expression ◽

Gene Promoter ◽

Mobility Shift ◽

Liver And Kidney ◽

Androgen Regulation ◽

Responsive Element ◽

Half Site

Cytosolic aspartate aminotransferase (cAspAT) is regulated by glucocorticoids in rat liver and kidney. Part of this regulation is mediated by an unusual glucocorticoid-responsive element (GRE)-like sequence called GRE A. GRE A is composed of two overlapping imperfect GREs, each comprising a conserved half-site (half-sites 1 and 4 respectively) and a poorly conserved half-site (half-sites 2 and 3 respectively). The sequence binds co-operatively two dimers of the glucocorticoid receptor (GR) and mediates efficient glucocorticoid regulation of gene expression. Analysis of deletions of the cAspAT gene promoter and subcloning of GRE A upstream of the thymidine kinase promoter indicate that this sequence is responsive to glucocorticoids, but not to androgens. Electrophoretic mobility shift assays indicate that the GRE A unit does not bind the androgen receptor (AR). The modification of three nucleotides in the poorly conserved half-sites 2 and 3, converting GRE A into two overlapping high-affinity GREs (ov-cGRE), resulted in co-operative binding of the AR. Furthermore, ov-cGRE efficiently mediated androgen regulation of the thymidine kinase promoter. A single base modification in half-site 2 or 3 in GRE A allowed the binding of the AR as one or two dimers respectively, and restored transcriptional activation by androgens only in the latter case. Thus the poor affinity of the AR for half-sites 2 and 3 prevented its binding to GRE A, indicating that the overlapping GRE A sequence of the cAspAT gene promoter discriminates a glucocorticoid-mediated from an androgen-mediated response.

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ATF4-Dependent Oxidative Induction of the DNA Repair Enzyme Ape1 Counteracts Arsenite Cytotoxicity and Suppresses Arsenite-Mediated Mutagenesis

Molecular and Cellular Biology ◽

10.1128/mcb.00974-07 ◽

2007 ◽

Vol 27 (24) ◽

pp. 8834-8847 ◽

Cited By ~ 33

Author(s):

Hua Fung ◽

Pingfang Liu ◽

Bruce Demple

Keyword(s):

Dna Repair ◽

Transcriptional Activation ◽

Activity Levels ◽

Cellular Mechanisms ◽

Mobility Shift ◽

Repair Enzyme ◽

Base Excision ◽

Transcription Suppression ◽

Tk6 Cells ◽

Electrophoretic Mobility Shift Assays

ABSTRACT Arsenite is a human carcinogen causing skin, bladder, and lung tumors, but the cellular mechanisms underlying these effects remain unclear. We investigated expression of the essential base excision DNA repair enzyme apurinic endonuclease 1 (Ape1) in response to sodium arsenite. In mouse 10T½ fibroblasts, Ape1 induction in response to arsenite occurred about equally at the mRNA, protein, and enzyme activity levels. Analysis of the APE1 promoter region revealed an AP-1/CREB binding site essential for arsenite-induced transcriptional activation in both mouse and human cells. Electrophoretic mobility shift assays indicated that an ATF4/c-Jun heterodimer was the responsible transcription factor. RNA interference targeting c-Jun or ATF4 eliminated arsenite-induced APE1 transcription. Suppression of Ape1 or ATF4 sensitized both mouse fibroblasts (10T½) and human lymphoblastoid cells (TK6) to arsenite cytotoxicity. Expression of Ape1 from a transgene did not efficiently restore arsenite resistance in ATF4-depleted cells but did offset initial accumulation of abasic DNA damage following arsenite treatment. Mutagenesis by arsenite (at the TK and HPRT loci in TK6 cells) was observed only for ATF4-depleted cells, which was strongly offset by Ape1 expression from a transgene. Therefore, the ATF4-mediated up-regulation of Ape1 and other genes plays a key role against arsenite-mediated toxicity and mutagenesis.

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Transcriptional activation of zebrafish cyp11a1 promoter is dependent on the nuclear receptor Ff1b

Journal of Molecular Endocrinology ◽

10.1677/jme-09-0029 ◽

2009 ◽

Vol 43 (3) ◽

pp. 121-130 ◽

Cited By ~ 6

Author(s):

Sue Ing Quek ◽

Woon Khiong Chan

Keyword(s):

Transcriptional Activation ◽

Bioinformatic Analysis ◽

Regulatory Elements ◽

Luciferase Reporter ◽

Egfp Expression ◽

Synthesis Rate ◽

Zebrafish Embryos ◽

Mobility Shift ◽

Cytochrome P450scc

The cytochrome P450scc (cholesterol side-chain cleavage enzyme) encoded by CYP11A1 catalyzes the first step in steroidogenesis by converting cholesterol to pregnenolone, and thus, controls the synthesis rate of steroid hormones. In mammals, steroidogenic factor 1 (SF1) has been implicated in the cAMP-mediated transcriptional activation of CYP11A1 promoter. In zebrafish, Ff1b has been established as the homolog of SF1. To assess the dependency of cyp11a1 expression on Ff1b, the putative promoter of zebrafish cyp11a1, spanning 1.7 kb, was isolated and bioinformatic analysis revealed two conserved FF1 response elements (FREs) that potentially bind Ff1b. Transfection studies in cell lines of different lineages confirmed that this promoter fragment contained the necessary regulatory elements required for its basal transcription. Truncation and mutagenesis studies performed in Y1 adrenocortical cells revealed that only the proximal FRE was essential for transcriptional activation. Electrophoretic mobility shift assay, however, indicated that Ff1b bound to both FREs, while their in vivo occupancy was confirmed using a chromatin immunoprecipitation assay. Lastly, the cyp11a1 promoter was able to direct EGFP expression specifically to the interrenal gland and genital ridge when transiently expressed in microinjected zebrafish embryos, and the promoter activity is potentiated by ff1b overexpression as measured from luciferase reporter activity in zebrafish embryos.

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Transcriptional Activation of Multiple Operons Involved inpara-Nitrophenol Degradation by Pseudomonas sp. Strain WBC-3

Applied and Environmental Microbiology ◽

10.1128/aem.02720-14 ◽

2014 ◽

Vol 81 (1) ◽

pp. 220-230 ◽

Cited By ~ 22

Author(s):

Wen-Mao Zhang ◽

Jun-Jie Zhang ◽

Xuan Jiang ◽

Hongjun Chao ◽

Ning-Yi Zhou

Keyword(s):

Transcriptional Activation ◽

Transcriptional Regulator ◽

Pseudomonas Sp ◽

Mobility Shift ◽

Content Type ◽

High Affinity ◽

Dnase I Footprinting ◽

Nitrophenol Degradation ◽

Transcriptional Start Sites ◽

Electrophoretic Mobility Shift Assays

ABSTRACTPseudomonassp. strain WBC-3 utilizespara-nitrophenol (PNP) as a sole carbon and energy source. The genes involved in PNP degradation are organized in the following three operons:pnpA,pnpB, andpnpCDEFG. How the expression of the genes is regulated is unknown. In this study, an LysR-type transcriptional regulator (LTTR) is identified to activate the expression of the genes in response to the specific inducer PNP. While the LTTR coding genepnpRwas found to be not physically linked to any of the three catabolic operons, it was shown to be essential for the growth of strain WBC-3 on PNP. Furthermore, PnpR positively regulated its own expression, which is different from the function of classical LTTRs. A regulatory binding site (RBS) with a 17-bp imperfect palindromic sequence (GTT-N11-AAC) was identified in allpnpA,pnpB,pnpC, andpnpRpromoters. Through electrophoretic mobility shift assays and mutagenic analyses, this motif was proven to be necessary for PnpR binding. This consensus motif is centered at positions approximately −55 bp relative to the four transcriptional start sites (TSSs). RBS integrity was required for both high-affinity PnpR binding and transcriptional activation ofpnpA,pnpB, andpnpR. However, this integrity was essential only for high-affinity PnpR binding to the promoter ofpnpCDEFGand not for its activation. Intriguingly, unlike other LTTRs studied, no changes in lengths of the PnpR binding regions of thepnpAandpnpBpromoters were observed after the addition of the inducer PNP in DNase I footprinting.

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Regulation of the Bacillus subtilis mannitol utilization genes: promoter structure and transcriptional activation by the wild-type regulator (MtlR) and its mutants

Microbiology ◽

10.1099/mic.0.071233-0 ◽

2014 ◽

Vol 160 (1) ◽

pp. 91-101 ◽

Cited By ~ 10

Author(s):

Kambiz Morabbi Heravi ◽

Josef Altenbuchner

Keyword(s):

Bacillus Subtilis ◽

Transcriptional Activation ◽

Transcription Initiation ◽

Phosphotransferase System ◽

Mobility Shift ◽

Dnase I Footprinting ◽

Proposed Model ◽

Rna Polymerase Holoenzyme ◽

Electrophoretic Mobility Shift Assays

Expression of mannitol utilization genes in Bacillus subtilis is directed by P mtlA , the promoter of the mtlAFD operon, and P mtlR , the promoter of the MtlR activator. MtlR contains phosphoenolpyruvate-dependent phosphotransferase system (PTS) regulation domains, called PRDs. The activity of PRD-containing MtlR is mainly regulated by the phosphorylation/dephosphorylation of its PRDII and EIIBGat-like domains. Replacing histidine 342 and cysteine 419 residues, which are the targets of phosphorylation in these two domains, by aspartate and alanine provided MtlR-H342D C419A, which permanently activates P mtlA in vivo. In the mtlR-H342D C419A mutant, P mtlA was active, even when the mtlAFD operon was deleted from the genome. The mtlR-H342D C419A allele was expressed in an Escherichia coli strain lacking enzyme I of the PTS. Electrophoretic mobility shift assays using purified MtlR-H342D C419A showed an interaction between the MtlR double-mutant and the Cy5-labelled P mtlA and P mtlR DNA fragments. These investigations indicate that the activated MtlR functions regardless of the presence of the mannitol-specific transporter (MtlA). This is in contrast to the proposed model in which the sequestration of MtlR by the MtlA transporter is necessary for the activity of MtlR. Additionally, DNase I footprinting, construction of P mtlA -P licB hybrid promoters, as well as increasing the distance between the MtlR operator and the −35 box of P mtlA revealed that the activated MtlR molecules and RNA polymerase holoenzyme likely form a class II type activation complex at P mtlA and P mtlR during transcription initiation.

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