scholarly journals A Functional Sp1 Binding Site Is Essential for the Activity of the Adult Liver-Specific Human Insulin-Like Growth Factor II Promoter

1997 ◽  
Vol 11 (2) ◽  
pp. 237-250 ◽  
Author(s):  
Richard J. T. Rodenburg ◽  
P. Elly Holthuizen ◽  
John S. Sussenbach
Keyword(s):  
Growth Factor ◽  
Binding Site ◽  
Insulin Like Growth Factor ◽  
Start Site ◽  
Adult Liver ◽  
Transcription Start ◽  
Mobility Shift ◽  
Enhancer Binding Protein ◽  
Factor Ii ◽  
Electrophoretic Mobility Shift Assays

Abstract The human gene encoding insulin-like growth factor II contains four promoters (P1–P4) that are differentially activated in various tissues during development. Expression of insulin-like growth factor II in adult liver tissue is directed by P1, which is activated by liver-enriched members of the CCAAT/enhancer binding protein family of transcription factors. In the present report we show that the region around −48 relative to the transcription start site contains a high affinity Sp1 binding site. This was demonstrated by electrophoretic mobility shift assays using nuclear extracts from Hep3B hepatoma cells and with specific antibodies directed against Sp1. Competition electrophoretic mobility shift assays revealed that the Sp1 binding site of P1 and a consensus Sp1 binding site bind Sp1 with comparable efficiencies. Mutation of the Sp1 binding site results in an 85% decrease in P1 promoter activity in transient transfection assays using two different cell lines, COS-7 and Hep3B. Investigation of P1 mutants in which the spacing of the Sp1 binding site and the transcription start site was increased showed that the role of the Sp1 binding site in regulation of P1 is position dependent. Interestingly, the Sp1-responsive element cannot be exchanged by a functional TATA box. Activation of P1 by transactivators CCAAT/enhancer binding protein-β and hepatocyte nuclear factor-3β is strongly impaired after mutation of the Sp1 binding site. These results demonstrate that the specific presence of a binding site for the ubiquitously expressed transcription factor Sp1 is of eminent importance for efficient activation of P1 by liver-enriched transactivators.

scholarly journals Growth-condition-dependent regulation of insulin-like growth factor II mRNA stability

1996 ◽  
Vol 318 (1) ◽  
pp. 195-201 ◽  
Author(s):  
Wiep SCHEPER ◽  
Elly HOLTHUIZEN ◽  
John S P. SUSSENBACH
Keyword(s):  
Growth Factor ◽  
De Novo ◽  
Mrna Level ◽  
Growth Conditions ◽  
Mrna Levels ◽  
Insulin Like Growth Factor ◽  
Mobility Shift ◽  
Main Site ◽  
Factor Ii ◽  
Hep3b Cells

Insulin-like growth factor II (IGF-II) is synthesized in many tissues, but the main site of production is the liver. In this paper we show that IGF-II mRNA levels are dependent on the growth conditions of the cells. In Hep3B cells, serum deprivation leads to a marked increase in IGF-II mRNA levels. Serum stimulation of starved Hep3B cells induces a decrease in the amount of IGF-II mRNA, which is not caused by a change in promoter activity. IGF-II mRNAs are subject to endonucleolytic cleavage, a process that requires two widely separated elements in the 3´ untranslated region of the mRNA. Specific regions of these elements can form a stable stem structure which is involved in the formation of RNA–protein complexes. By employing electrophoretic mobility shift assays, two complexes have been identified in cytoplasmic extracts of Hep3B cells. The formation of these complexes is related to the growth conditions of the cells and is correlated with the regulation of IGF-II mRNA levels. Our data suggest that, depending on whether serum is present or absent, a transition from one complex to the other occurs. A decrease in the IGF-II mRNA level is also observed when IGF-I or IGF-II is added to serum-deprived Hep3B cells, possibly providing a feedback mechanism for IGF-II production. The serum-induced degradation of IGF-II mRNAs does not require de novo protein synthesis, and is abolished by rapamycin, an inhibitor of p70 S6 kinase.

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 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 A Novel Binding Site for the Human Insulin-like Growth Factor-II (IGF-II)/Mannose 6-Phosphate Receptor on IGF-II

2007 ◽  
Vol 282 (26) ◽  
pp. 18886-18894 ◽  
Author(s):  
Carlie Delaine ◽  
Clair L. Alvino ◽  
Kerrie A. McNeil ◽  
Terrance D. Mulhern ◽  
Lisbeth Gauguin ◽  
...  
Keyword(s):  
Growth Factor ◽  
Binding Site ◽  
Human Insulin ◽  
Insulin Like Growth Factor ◽  
Factor Ii ◽  
Mannose 6 Phosphate
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