scholarly journals Sp1 and Sp3 regulate glucokinase gene transcription in the liver of gilthead sea bream (Sparus aurata)

2007 ◽  
Vol 38 (4) ◽  
pp. 481-492 ◽  
Author(s):  
M Egea ◽  
I Metón ◽  
I V Baanante
Keyword(s):  
Binding Site ◽  
Transcription Start Site ◽  
Hepg2 Cells ◽  
Sparus Aurata ◽  
Gilthead Sea Bream ◽  
Sea Bream ◽  
Proximal Promoter ◽  
Start Site ◽  
Transcription Start ◽  
Mobility Shift

To better understand the transcriptional machinery that governs glucokinase (GCK) expression, we have cloned and characterized the proximal promoter region of GCK from gilthead sea bream (Sparus aurata). The 5′-flanking region of GCK was isolated by chromosome walking. SMART RACE-PCR allowed us to locate the transcription start site 98 bp (bp) upstream from the translational start. Transfection analysis in HepG2 cells revealed the presence of a functional promoter in the 1397 bp 5′-flanking isolated fragment (positions −1321 to +76 relative to the transcription start site). Sequential 5′-deletion analysis indicated a core functional promoter for basal transcription within the 288 bp upstream from the transcription start site. Transient transfection experiments performed in HepG2 cells and electrophoretic mobility shift assays denoted that Sp1 binds and transactivates GCK promoter, whereas Sp3 repressed Sp1-mediated activation of GCK by competing for the same binding site. Mutations in the Sp binding site completely abolished the enhancing effect of Sp1. Treatment with insulin stimulated GCK expression, and increased Sp1 levels in S. aurata liver. We propose a new mechanism that involves Sp1 and Sp3 to mediate insulin activation of GCK transcription.

scholarly journals Characterization of Enhancer Binding by the Vibrio cholerae Flagellar Regulatory Protein FlrC

2005 ◽  
Vol 187 (9) ◽  
pp. 3158-3170 ◽  
Author(s):  
Nidia E. Correa ◽  
Karl E. Klose
Keyword(s):  
Binding Site ◽  
Vibrio Cholerae ◽  
Transcription Start Site ◽  
Regulatory Protein ◽  
Dnase I ◽  
Start Site ◽  
Transcription Start ◽  
Mobility Shift ◽  
Enhancer Element ◽  
Dnase I Footprinting

ABSTRACT The human pathogen Vibrio cholerae is a highly motile organism by virtue of a polar flagellum, and motility has been inferred to be an important aspect of virulence. It has previously been demonstrated that the σ54-dependent activator FlrC is necessary for both flagellar synthesis and for enhanced intestinal colonization. In order to characterize FlrC binding, we analyzed two FlrC-dependent promoters, the highly transcribed flaA promoter and the weakly transcribed flgK promoter, utilizing transcriptional lacZ fusions, mobility shift assays, and DNase I footprinting. Promoter fusion studies showed that the smallest fragment with wild-type transcriptional activity for flaAp was from positions −54 to +137 with respect to the start site, and from −63 to +144 for flgKp. Gel mobility shift assays indicated that FlrC binds to a fragment containing the region from positions +24 to +95 in the flaAp, and DNase I footprinting identified a protected region between positions +24 and +85. Mobility shift and DNase I footprinting indicated weak binding of FlrC to a region downstream of the flgKp transcription start site. These results demonstrate a relatively novel σ54-dependent promoter architecture, with the activator FlrC binding downstream of the σ54-dependent transcription start sites. When the FlrC binding site(s) in the flaA promoter was moved a large distance (285 bp) upstream of the transcription start site of either flaAp or flgKp, high levels of FlrC-dependent transcription resulted, indicating that this binding region functions as an enhancer element. In contrast, the relatively weak FlrC binding site(s) in the flgK promoter failed to function as an enhancer element at either promoter, suggesting that FlrC binding strength contributes to enhancer activity. Our results suggest that the differences in FlrC binding to various flagellar promoters results in the differences in transcription levels that mirror the relative requirement for the flagellar components within the flagellum.

scholarly journals Co-expression of IGFs and GH receptors (GHRs) in gilthead sea bream (Sparus aurata L.): sequence analysis of the GHR-flanking region

2007 ◽  
Vol 194 (2) ◽  
pp. 361-372 ◽  
Author(s):  
Alfonso Saera-Vila ◽  
Josep Alvar Calduch-Giner ◽  
Jaume Pérez-Sánchez
Keyword(s):  
Adipose Tissue ◽  
Transcription Start Site ◽  
Sparus Aurata ◽  
Juvenile Fish ◽  
Sea Bream ◽  
Start Site ◽  
Transcription Start ◽  
Igf I ◽  
Functional Partitioning ◽  
Gh Receptors

The tissue-specific expression of IGFs and GH receptors (GHRs) was analyzed in gilthead sea bream (Sparus aurata L.) as an attempt to understand the functional partitioning of duplicated GHRs on the regulation offish growth by season and aging. Gene transcripts were measured in liver, muscle, and adipose tissue by means of quantitative real-time PCR assays. In juvenile fish, concurrent increases in circulating levels of GH and IGF-I and hepatic mRNA levels of IGF-I and GHR-I were evidenced with the summer growth spurt. Conversely, muscle and adipose tissue expression of GHR-I and IGF-II were significantly upregulated by over wintering. The aging decrease of growth rates was accompanied by a reduced activity of the liver GH/IGF axis, and parallel increases in muscle IGF expression would be dictated at the local tissue level by the enhanced expression of GHR-I. Extra-hepatic expression of IGFs and GHR-II did not correlate seasonally in juvenile fish, and nonsignificant effects of aging were found on the summer expression of GHR-II in any analyzed tissue. One transcription start site was identified by RLM-RACE in GHR-I and GHR-II. Sequence analyses indicated that both genes have TATA-less promoters containing consensus initiator sequences and downstream promoter elements surrounding the transcription start site. Conserved CCAAT-boxes and GC-rich regions were retrieved in the GHR-I promoter, whereas stress- and redox-sequence elements (cAMP-responsive element-binding protein, activator proteins; AP-1, and AP-4) were characteristic features of GHR-II. All this supports the functional partitioning of fish GHRs regardless of fish species differences.

scholarly journals Transcriptional regulation of glucose-6-phosphatase catalytic subunit promoter by insulin and glucose in the carnivorous fish, Sparus aurata

2004 ◽  
Vol 33 (3) ◽  
pp. 783-795 ◽  
Author(s):  
M C Salgado ◽  
I Metón ◽  
M Egea ◽  
I V Baanante
Keyword(s):  
Transcription Start Site ◽  
Hepg2 Cells ◽  
Sparus Aurata ◽  
Catalytic Subunit ◽  
Mrna Levels ◽  
Start Site ◽  
Transcription Start ◽  
Carnivorous Fish ◽  
Repressive Effect ◽  
Insulin Dependent

Increase in glucose-6-phosphatase catalytic subunit (G6Pase, G6pc) transcription enhances hepatic glucose production in non-insulin-dependent diabetes mellitus (NIDDM). The fact that carnivorous fish is an alternative model to study NIDDM led us to clone and characterise the first G6pc promoter region reported for fish and non-mammalian animals. The 5′-flanking region of G6pc from gilthead sea bream (Sparus aurata) was isolated by chromosome walking. With SMART RACE-PCR, the transcription start site was located 106 base pairs (bp) upstream of the translational start. Transfection analysis in HepG2 cells located a functional promoter in the 850 bp 5′-flanking isolated fragment (positions −770 to +80 relative to the transcription start). Sequential 5′-deletion analysis of the promoter fragment revealed that a core functional promoter for basal transcription is comprised within the 190 bp upstream of the transcription start site. In vivo, glucose and insulin reduced G6Pase mRNA levels in the fish liver. Transfection experiments in HepG2 cells showed that insulin repressed S. aurata G6pc under high-glucose conditions. Synergistic activation of piscine G6pc promoter was induced by cotransfection with expression plasmids for hepatocyte nuclear factor-4α (HNF-4α) and peroxisome proliferator-activated receptor-γ coactivator-1 (PGC-1α). No direct relationship was found between PGC-1α coactivation of HNF-4α transactivation and the repressive effect of insulin. Interestingly, insulin hardly affected G6pc promoter activity in the absence of glucose, suggesting that a reduced capacity of insulin-dependent repression of piscine G6pc may lead to insulin resistance in carnivorous fish.

scholarly journals Transcription Regulation by Tandem-Bound FNR at Escherichia coli Promoters

2003 ◽  
Vol 185 (20) ◽  
pp. 5993-6004 ◽  
Author(s):  
Anne M. L. Barnard ◽  
Jeffrey Green ◽  
Stephen J. W. Busby
Keyword(s):  
Escherichia Coli ◽  
Transcription Factor ◽  
Binding Site ◽  
Transcription Regulation ◽  
Transcription Start Site ◽  
Promoter Activity ◽  
Start Site ◽  
Transcription Start ◽  
Substitution Mutant

ABSTRACT FNR is an Escherichia coli transcription factor that regulates the transcription of many genes in response to anaerobiosis. We have constructed a series of artificial FNR-dependent promoters, based on the melR promoter, in which a consensus FNR binding site was centered at position −41.5 relative to the transcription start site. A second consensus FNR binding site was introduced at different upstream locations, and promoter activity was assayed in vivo. FNR can activate transcription from these promoters when the upstream FNR binding site is located at many different positions. However, sharp repression is observed when the upstream-bound FNR is located near positions −85 or −95. This repression is relieved by the FNR G74C substitution mutant, previously identified as being defective in transcription repression at the yfiD promoter. A parallel series of artificial FNR-dependent promoters, carrying a consensus FNR binding site at position −61.5 and a second upstream DNA site for FNR, was also constructed. Again, promoter activity was repressed by FNR when the upstream-bound FNR was located at particular positions.

scholarly journals Characterization of the human proline/arginine-rich end leucine-rich repeat protein (PRELP) gene promoter and identification of a repressor element

1998 ◽  
Vol 336 (1) ◽  
pp. 77-82 ◽  
Author(s):  
Judy GROVER ◽  
Peter J. ROUGHLEY
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Transcription Start Site ◽  
Gene Promoter ◽  
Start Site ◽  
Leucine Rich Repeat ◽  
Transcription Start ◽  
Repeat Protein ◽  
Repressor Element ◽  
Leucine Rich Repeat Protein

The 5´-flanking region of the human proline/arginine-rich end leucine-rich repeat protein (PRELP) gene has been characterized for both promoter and repressor activity by using a variety of reporter gene constructs and transient transfection into chondrocytes or fibroblasts. The human PRELP gene lacks a TATA box, and in its absence a Sp1-binding site residing 29 bp upstream of the transcription start site is essential for initiating gene expression. In contrast, an Ets-binding site residing 497 bp upstream of the transcription start site can lead to the repression of gene expression. The analysis of nuclear proteins by gel retardation studies with the repressor element identified a common protein, presumably an Ets family member, present in neonatal chondrocytes and skin fibroblasts that do not express the PRELP gene. The factor was not detected in nuclear protein preparations from adult chondrocytes in which the PRELP gene is expressed.

scholarly journals Regulation of the EDG84A gene by FTZ-F1 during metamorphosis in Drosophila melanogaster.

1996 ◽  
Vol 16 (11) ◽  
pp. 6509-6515 ◽  
Author(s):  
T Murata ◽  
Y Kageyama ◽  
S Hirose ◽  
H Ueda
Keyword(s):  
Drosophila Melanogaster ◽  
Binding Site ◽  
Transcription Start Site ◽  
Fusion Gene ◽  
Regulatory Region ◽  
Posterior Part ◽  
Nuclear Hormone Receptor ◽  
The Body ◽  
Start Site ◽  
Transcription Start

The transcription factor FTZ-F1 is a member of the nuclear hormone receptor superfamily and is transiently expressed during the mid- and late prepupal periods in Drosophila melanogaster. A putative pupal cuticle gene, EDG84A, is expressed slightly following FTZ-F1 expression during the prepupal period and carries a strong FTZ-F1 binding site between bases 100 and 92 upstream of its transcription start site. In this study, EDG84A mRNA was found to be prematurely expressed upon heat induction of FTZ-F1 in prepupae carrying the heat shock promoter-FTZ-F1 cDNA fusion gene construct. Transgenic fly lines having the 0.8-kb region of the EDG84A promoter fused to lacZ expressed the reporter gene in a tissue- and stage-specific manner. Base substitutions in the FTZ-F1 binding site within the 0.8-kb promoter abolished expression of lacZ. These results strongly suggest that the EDG84A gene is a direct target of FTZ-F1. Deletion studies of the cis-regulatory region of the EDG84A gene revealed that space-specific expression in imaginal disc-derived epidermis is controlled by the region between bp -408 and -104 from the transcription start site. The region between bp -408 and -194 is necessary to repress expression in a posterior part of the body, while the region between bp -193 and -104 carries a positive element for activation in an anterior part of the body. These results suggest that FTZ-F1 governs expression of the EDG84A gene in conjunction with putative tissue-specific regulators.

scholarly journals Transcriptional promoter of the human α1(V) collagen gene (COL5A1)

1995 ◽  
Vol 310 (1) ◽  
pp. 15-22 ◽  
Author(s):  
S Lee ◽  
D S Greenspan
Keyword(s):  
Transcription Start Site ◽  
Cpg Island ◽  
Growth Control ◽  
Gc Content ◽  
Collagen Gene ◽  
Start Site ◽  
Transcription Start ◽  
Mobility Shift ◽  
Broad Distribution ◽  
S1 Nuclease

We have characterized the 5′ region of the human alpha 1(V) collagen gene (COL5A1). The transcriptional promoter is shown to have a number of features characteristic of the promoters of ‘housekeeping’ and growth-control-related genes. It lacks obvious TATA and CAAT boxes, has multiple transcription start sites, has a high GC content, lies within a well-defined CpG island and has a number of consensus sites for the potential binding of transcription factor Sp1. This type of promoter structure, while unusual for a collagen gene, is consistent with the broad distribution of expression of COL5A1 and is reminiscent of the promoter structures of the genes encoding type VI collagen, which has a similarly broad distribution of expression. Stepwise deletion of COL5A1 5′ sequences, placed upstream of a heterologous reporter gene, yielded a gradual decrease in promoter activity, indicating that the COL5A1 promoter is composed of an array of cis-acting elements. A minimal promoter region contained within the 212 bp immediately upstream of the major transcription start site contained no consensus sequences for the binding of known transcription factors, but gel mobility shift assays showed this region to bind nuclear factors, including Sp1, at a number of sites. The major transcription start site is flanked by an upstream 34-bp oligopurine/oligopyrimidine stretch, or ‘GAGA’ box, and a downstream 56-bp GAGA box which contains a 10-bp mirror repeat and is sensitive to cleavage with S1 nuclease.

scholarly journals Location of promoter elements necessary and sufficient to direct testis-specific expression of the Hst70/Hsp70.2 gene

2004 ◽  
Vol 379 (3) ◽  
pp. 739-747 ◽  
Author(s):  
Dorota ŚCIEGLIŃSKA ◽  
Natallia VYDRA ◽  
Zdzisław KRAWCZYK ◽  
Wiesława WIDŁAK
Keyword(s):  
Gene Expression ◽  
Transcription Start Site ◽  
Gene Promoter ◽  
Specific Gene ◽  
Start Site ◽  
Transcription Start ◽  
Mobility Shift ◽  
Specific Expression ◽  
High Level ◽  
Testis Specific Expression

The rat Hst70 gene and its mouse counterpart Hsp70.2 are expressed specifically in pachytene primary spermatocytes and spermatids. Here we demonstrate that a 165 bp fragment of the Hst70 gene promoter, containing the T1 transcription start site region, entire exon 1 and 42 bp 5´ region of the intron, is sufficient to drive testis-specific expression of the chloramphenicol acetyltransferase reporter gene in transgenic mice with the same developmentally regulated pattern as the endogenous Hsp70.2 gene. We show further that high-level tissue-specific gene expression requires additional sequences localized upstream of the T2 transcription start site. Electrophoretic mobility-shift assay analysis revealed that only testes of juvenile rats, when Hst70 gene expression is repressed, contain proteins that specifically bind to the Oct (octamer) sequence localized directly downstream of the T1 site.

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