scholarly journals Pi class glutathione S-transferase genes are regulated by Nrf 2 through an evolutionarily conserved regulatory element in zebrafish

2005 ◽  
Vol 388 (1) ◽  
pp. 65-73 ◽  
Author(s):  
Takafumi SUZUKI ◽  
Yaeko TAKAGI ◽  
Hitoshi OSANAI ◽  
Li LI ◽  
Miki TAKEUCHI ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Fluorescent Protein ◽  
Regulatory Element ◽  
Initiation Site ◽  
Genomic Region ◽  
Related Factor ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Mobility Shift Assay ◽  
Green Fluorescent

Pi class GSTs (glutathione S-transferases) are a member of the vertebrate GST family of proteins that catalyse the conjugation of GSH to electrophilic compounds. The expression of Pi class GST genes can be induced by exposure to electrophiles. We demonstrated previously that the transcription factor Nrf 2 (NF-E2 p45-related factor 2) mediates this induction, not only in mammals, but also in fish. In the present study, we have isolated the genomic region of zebrafish containing the genes gstp1 and gstp2. The regulatory regions of zebrafish gstp1 and gstp2 have been examined by GFP (green fluorescent protein)-reporter gene analyses using microinjection into zebrafish embryos. Deletion and point-mutation analyses of the gstp1 promoter showed that an ARE (antioxidant-responsive element)-like sequence is located 50 bp upstream of the transcription initiation site which is essential for Nrf 2 transactivation. Using EMSA (electrophoretic mobility-shift assay) analysis we showed that zebrafish Nrf 2–MafK heterodimer specifically bound to this sequence. All the vertebrate Pi class GST genes harbour a similar ARE-like sequence in their promoter regions. We propose that this sequence is a conserved target site for Nrf 2 in the Pi class GST genes.

scholarly journals Isolation and characterization of the androgen-dependent mouse cysteine-rich secretory protein-1 (CRISP-1) gene

1997 ◽  
Vol 321 (2) ◽  
pp. 325-332 ◽  
Author(s):  
Uta SCHWIDETZKY ◽  
Wolf-Dieter SCHLEUNING ◽  
Bernard HAENDLER
Keyword(s):  
Transcription Initiation ◽  
Initiation Site ◽  
Secretory Protein ◽  
Mobility Shift ◽  
Isolation And Characterization ◽  
Eukaryotic Genes ◽  
Mobility Shift Assay ◽  
Responsive Element

In mice, cysteine-rich secretory protein-1 (CRISP-1) is mainly found in the epididymis and also, to a lesser extent, in the salivary gland of males, where androgens control its expression. We have now isolated and characterized overlapping phage clones covering the entire length of the CRISP-1gene. DNA sequencing revealed that the gene is organized into eight exons, ranging between 55 and 748 bp in size, and seven introns. All exonŐintron junctions conformed to the GT/AG rule established for eukaryotic genes. The intron length, as determined by PCR, varied between 1.05 and 4.0 kb so that the CRISP-1gene spans over 20 kb of the mouse genome. The transcription-initiation site was determined by primer extension and localized at the expected distance downstream of a consensus TATA box. Approximately 3.7 kb of the CRISP-1promoter region were isolated and sequenced, and several stretches fitting the androgen-responsive element consensus were found. Those that most resembled the consensus were analysed by electrophoretic mobility-shift assay and found to form specific complexes with the liganded androgen receptor in vitro, but with different affinities. Putative binding elements for the transcription factors Oct, GATA, PEA3, CF1, AP-1 and AP-3 were also found in the promoter region.

scholarly journals Combination of Two Regulatory Elements in the Tetrahymena thermophila HSP70-1 Gene Controls Heat Shock Activation

2007 ◽  
Vol 7 (2) ◽  
pp. 379-386 ◽  
Author(s):  
Sabrina Barchetta ◽  
Antonietta La Terza ◽  
Patrizia Ballarini ◽  
Sandra Pucciarelli ◽  
Cristina Miceli
Keyword(s):  
Heat Shock ◽  
Fluorescent Protein ◽  
Tetrahymena Thermophila ◽  
Regulatory Elements ◽  
Mobility Shift ◽  
Functional Link ◽  
In Vivo Analysis ◽  
Mobility Shift Assay ◽  
Green Fluorescent

ABSTRACT The induction of heat shock genes (HSPs) is thought to be primarily regulated by heat shock transcription factors (HSFs), which bind target sequences on HSP promoters, called heat shock elements (HSEs). In this study, we investigated the 5′ untranslated regions of the Tetrahymena thermophila HSP70-1 gene, and we found, in addition to the canonical and divergent HSEs, multiple sets of GATA elements that have not been reported previously in protozoa. By means of in vivo analysis of a green fluorescent protein reporter transgene driven by the HSP70-1 promoter, we demonstrate that HSEs do not represent the minimal regulatory elements for heat shock induction, since the HSP70-1 is tightly regulated by both HSE and GATA elements. Electrophoretic mobility shift assay also showed that HSFs are constitutively bound to the HSEs, whereas GATA elements are engaged only after heat shock. This is the first demonstration by in vivo analysis of functional HSE and GATA elements in protozoa. Furthermore, we provide evidence of a functional link between HSE and GATA elements in the activation of the heat shock response.

Identification of upstream and intragenic regulatory elements that confer cell-type-restricted and differentiation-specific expression on the muscle creatine kinase gene

1988 ◽  
Vol 8 (7) ◽  
pp. 2896-2909 ◽  
Author(s):  
E A Sternberg ◽  
G Spizz ◽  
W M Perry ◽  
D Vizard ◽  
T Weil ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Regulatory Element ◽  
Simian Virus 40 ◽  
Muscle Cells ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Simian Virus ◽  
Cell Type ◽  
Specific Expression ◽  
Developing Muscle

Terminal differentiation of skeletal myoblasts is accompanied by induction of a series of tissue-specific gene products, which includes the muscle isoenzyme of creatine kinase (MCK). To begin to define the sequences and signals involved in MCK regulation in developing muscle cells, the mouse MCK gene has been isolated. Sequence analysis of 4,147 bases of DNA surrounding the transcription initiation site revealed several interesting structural features, some of which are common to other muscle-specific genes and to cellular and viral enhancers. To test for sequences required for regulated expression, a region upstream of the MCK gene from -4800 to +1 base pairs, relative to the transcription initiation site, was linked to the coding sequences of the bacterial chloramphenicol acetyltransferase (CAT) gene. Introduction of this MCK-CAT fusion gene into C2 muscle cells resulted in high-level expression of CAT activity in differentiated myotubes and no detectable expression in proliferating undifferentiated myoblasts or in nonmyogenic cell lines. Deletion mutagenesis of sequences between -4800 and the transcription start site showed that the region between -1351 and -1050 was sufficient to confer cell type-specific and developmentally regulated expression on the MCK promoter. This upstream regulatory element functioned independently of position, orientation, or distance from the promoter and therefore exhibited the properties of a classical enhancer. This upstream enhancer also was able to confer muscle-specific regulation on the simian virus 40 promoter, although it exhibited a 3- to 5-fold preference for its own promoter. In contrast to the cell type- and differentiation-specific expression of the upstream enhancer, the MCK promoter was able to function in myoblasts and myotubes and in nonmyogenic cell lines when combined with the simian virus 40 enhancer. An additional positive regulatory element was identified within the first intron of the MCK gene. Like the upstream enhancer, this intragenic element functioned independently of position, orientation, and distance with respect to the MCK promoter and was active in differentiated myotubes but not in myoblasts. These results demonstrate that expression of the MCK gene in developing muscle cells is controlled by complex interactions among multiple upstream and intragenic regulatory elements that are functional only in the appropriate cellular context.

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.

scholarly journals Transcription of Drosophila Troponin I Gene Is Regulated by Two Conserved, Functionally Identical, Synergistic Elements

2004 ◽  
Vol 15 (3) ◽  
pp. 1185-1196 ◽  
Author(s):  
María-Cruz Marín ◽  
José-Rodrigo Rodríguez ◽  
Alberto Ferrús
Keyword(s):  
Transcription Initiation ◽  
Troponin I ◽  
Regulatory Element ◽  
Expression Patterns ◽  
In Silico Analysis ◽  
Initiation Site ◽  
Transcription Initiation Site ◽  
Upstream Regulatory Element ◽  
Transgenic Lines

The Drosophila wings-up A gene encodes Troponin I. Two regions, located upstream of the transcription initiation site (upstream regulatory element) and in the first intron (intron regulatory element), regulate gene expression in specific developmental and muscle type domains. Based on LacZ reporter expression in transgenic lines, upstream regulatory element and intron regulatory element yield identical expression patterns. Both elements are required for full expression levels in vivo as indicated by quantitative reverse transcription-polymerase chain reaction assays. Three myocyte enhancer factor-2 binding sites have been functionally characterized in each regulatory element. Using exon specific probes, we show that transvection is based on transcriptional changes in the homologous chromosome and that Zeste and Suppressor of Zeste 3 gene products act as repressors for wings-up A. Critical regions for transvection and for Zeste effects are defined near the transcription initiation site. After in silico analysis in insects (Anopheles and Drosophila pseudoobscura) and vertebrates (Ratus and Coturnix), the regulatory organization of Drosophila seems to be conserved. Troponin I (TnI) is expressed before muscle progenitors begin to fuse, and sarcomere morphogenesis is affected by TnI depletion as Z discs fail to form, revealing a novel developmental role for the protein or its transcripts. Also, abnormal stoichiometry among TnI isoforms, rather than their absolute levels, seems to cause the functional muscle defects.

scholarly journals Transcriptional and Functional Analysis of the Neisseria gonorrhoeae Fur Regulon

2009 ◽  
Vol 192 (1) ◽  
pp. 77-85 ◽  
Author(s):  
Lydgia A. Jackson ◽  
Thomas F. Ducey ◽  
Michael W. Day ◽  
Jeremy B. Zaitshik ◽  
Joshua Orvis ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Transcriptional Control ◽  
Cell Communication ◽  
Essential Element ◽  
Open Reading Frames ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Sequence Logos ◽  
Mobility Shift Assay

ABSTRACT To ensure survival in the host, bacteria have evolved strategies to acquire the essential element iron. In Neisseria gonorrhoeae, the ferric uptake regulator Fur regulates metabolism through transcriptional control of iron-responsive genes by binding conserved Fur box (FB) sequences in promoters during iron-replete growth. Our previous studies showed that Fur also controls the transcription of secondary regulators that may, in turn, control pathways important to pathogenesis, indicating an indirect role for Fur in controlling these downstream genes. To better define the iron-regulated cascade of transcriptional control, we combined three global strategies—temporal transcriptome analysis, genomewide in silico FB prediction, and Fur titration assays (FURTA)—to detect genomic regions able to bind Fur in vivo. The majority of the 300 iron-repressed genes were predicted to be of unknown function, followed by genes involved in iron metabolism, cell communication, and intermediary metabolism. The 107 iron-induced genes encoded hypothetical proteins or energy metabolism functions. We found 28 predicted FBs in FURTA-positive clones in the promoters and within the open reading frames of iron-repressed genes. We found lower levels of conservation at critical thymidine residues involved in Fur binding in the FB sequence logos of FURTA-positive clones with intragenic FBs than in the sequence logos generated from FURTA-positive promoter regions. In electrophoretic mobility shift assay studies, intragenic FBs bound Fur with a lower affinity than intergenic FBs. Our findings further indicate that transcription under iron stress is indirectly controlled by Fur through 12 potential secondary regulators.

scholarly journals Genomic organization and transcriptional analysis of the human l-glutaminase gene

2003 ◽  
Vol 370 (3) ◽  
pp. 771-784 ◽  
Author(s):  
Cristina PÉREZ-GÓMEZ ◽  
José M. MATÉS ◽  
Pedro M. GÓMEZ-FABRE ◽  
Antonio del CASTILLO-OLIVARES ◽  
Francisco J. ALONSO ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Genomic Organization ◽  
Core Promoter ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Genome Project ◽  
Transcriptional Analysis ◽  
Cdna Clones ◽  
Mobility Shift ◽  
Specific Expression

In mammals, glutaminase (GA) is expressed in most tissues, but the regulation of organ-specific expression is largely unknown. Therefore, as an essential step towards studying the regulation of GA expression, the human liver-type GA (hLGA) gene has been characterized. LGA genomic sequences were isolated using the genome walking technique. Analysis and comparison of these sequences with two LGA cDNA clones and the Human Genome Project database, allowed the determination of the genomic organization of the LGA gene. The gene has 18 exons and is approx. 18kb long. All exon/intron junction sequences conform to the GT/AG rule. Progressive deletion analysis of LGA promoter—luciferase constructs indicated that the core promoter is located between nt −141 and +410, with several potential regulatory elements: CAAT, GC, TATA-like, Ras-responsive element binding protein and specificity protein 1 (Sp1) sites. The minimal promoter was mapped within +107 and +410, where only an Sp1 binding site is present. Mutation experiments suggested that two CAAT recognition elements near the transcription-initiation site (-138 and −87), play a crucial role for optimal promoter activity. Electrophoretic mobility-shift assays confirmed the importance of CAAT- and TATA-like boxes to enhance basal transcription, and demonstrated that HNF-1 motif is a significant distal element for transcriptional regulation of the hLGA gene.

scholarly journals Transcribing paramyxovirus RNA polymerase engages the template at its 3′ extremity

2006 ◽  
Vol 87 (3) ◽  
pp. 665-672 ◽  
Author(s):  
Samuel Cordey ◽  
Laurent Roux
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Fluorescent Protein ◽  
Sendai Virus ◽  
Rna Viruses ◽  
Mrna Transcription ◽  
Gfp Expression ◽  
Rnase Protection ◽  
Virus Rna ◽  
Green Fluorescent

For the non-segmented, negative-stranded RNA viruses, the mechanism controlling transcription or replication is still a matter of debate. To gain information about this mechanism and about the nature of the RNA polymerase involved, the length of an intervening sequence separating the 3′ end of Sendai virus minigenomes and a downstream transcription-initiation signal was increased progressively. It was found that transcription, as measured by green fluorescent protein (GFP) expression, decreased progressively in proportion to the increase in length of the intervening sequence. GFP expression correlated well with the levels of GFP mRNA in the cells, as measured by quantitative primer extension and by RNase protection. Thus, mRNA transcription was inversely proportional to the length of the inserted sequence. These data are evidence that the RNA polymerase initiating transcription at the downstream transcription signal somehow sees the distance separating this signal and the template 3′ extremity. Implication of this observation for the nature of the Sendai virus RNA polymerase and for the mechanism by which it synthesizes mRNAs or replication products is presented.

scholarly journals Transcriptional regulation of human and murine 17β-hydroxysteroid dehydrogenase type-7 confers its participation in cholesterol biosynthesis

2006 ◽  
Vol 37 (1) ◽  
pp. 185-197 ◽  
Author(s):  
Thomas Ohnesorg ◽  
Brigitte Keller ◽  
Martin Hrabé de Angelis ◽  
Jerzy Adamski
Keyword(s):  
Transcriptional Regulation ◽  
Binding Sites ◽  
Sequence Similarity ◽  
Regulatory Element ◽  
Cholesterol Biosynthesis ◽  
Hydroxysteroid Dehydrogenase ◽  
Site Directed Mutagenesis ◽  
Mobility Shift ◽  
Mobility Shift Assay

In both humans and mice, 17β-hydroxysteroid dehydrogenase type-7 (HSD17B7) was described as possessing dual enzymatic functionality. The enzyme was first shown to be able to convert estrone to estradiol in vitro. Later involvement of this enzyme in postsqualene cholesterol biosynthesis was postulated (conversion of zymosterone to zymosterol) and could be proven in vitro. In this work, we performed a detailed analysis of the transcriptional regulation of both the human and murine genes. Despite relatively low sequence similarity, both promoters contain similar contexts of transcription factor-binding sites. The participation of these sites in transcriptional regulation of HSD17B7 was proven by electro-mobility shift assay and site-directed mutagenesis of the corresponding binding sites. We describe novel involvement of vitamin D receptor/retinoid X receptor and provide new information on the regulation of HSD17B7 expression by sterol regulatory element-binding protein and hepatocyte nuclear factor 4, the latter known from other genes of cholesterogenic enzymes. The results of our study provide unequivocal evidence for a role of HSD17B7 in cholesterol biosynthesis.

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