scholarly journals Gene expression of the dibasic-pair cleaving enzyme NRD convertase (N-arginine dibasic convertase) is differentially regulated in the GH3 pituitary and Mat-Lu prostate cell lines

2000 ◽  
Vol 351 (3) ◽  
pp. 755-764
Author(s):  
Andrew G. WINTER ◽  
Adrian R. PIEROTTI
Keyword(s):  
Transcription Factor ◽  
Electrophoretic Mobility ◽  
Cell Lines ◽  
Binding Sites ◽  
Transcription Factor Binding Sites ◽  
Transcription Factor Binding ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Factor Binding ◽  
Electrophoretic Mobility Shift Assays

NRD convertase (N-arginine dibasic convertase, NRD-C) is a dibasic selective metalloprotease which cleaves on the N-terminal side of an arginine residue in a dibasic pair. Abundant in endocrine tissues, the highest levels are found in testis. The mechanism whereby NRD-C expression is regulated at the transcriptional level has been examined by reporter-gene assay and electrophoretic-mobility-shift assays. Analysis of the rat and human promoters show that they are highly conserved, containing a number of motifs which may correspond to transcription-factor binding sites. The rat promoter has been cloned into a luciferase reporter vector and analysed in a number of cell lines. Full functionality of the promoter is observed with 5′ deletions to 411bp upstream of the transcriptional start site in spermatid, prostate and pituitary cell lines. Further deletion to 101bp causes a complete loss of activity in spermatid and prostate lines. By contrast, GH3 pituitary cells display no reduction in promoter activity with deletion to 101bp of upstream sequence. A number of transcription-factor binding sites have been identified by electrophoretic-mobility-shift assays in the region 411–101; however, no differences in binding between the cell lines were observed.

Identification of a novel region in the proximal promoter of the mouse renin gene critical for expression

2004 ◽  
Vol 286 (6) ◽  
pp. F1107-F1115 ◽  
Author(s):  
Li Pan ◽  
Craig A. Jones ◽  
Sean T. Glenn ◽  
Kenneth W. Gross
Keyword(s):  
Transcription Factor ◽  
Binding Site ◽  
Electrophoretic Mobility ◽  
Binding Sites ◽  
Promoter Activity ◽  
Transcription Factor Binding Sites ◽  
Proximal Promoter ◽  
Mobility Shift ◽  
Factor Binding ◽  
Renin Gene

An enhancer at −2.6 kb and a HOX·PBX-binding site at −60 bp have been demonstrated to be critical to expression of the mouse renin gene ( Ren-1 c) in As4.1 cells. In this report, we show that a region (−197 to −70) immediately 5′ to the HOX·PBX-binding site is also critical for Ren-1 c expression. Deletion of this region in a construct containing 4.1 kb of the Ren-1 c 5′-flanking sequence resulted in a 99% reduction in Ren-1 c promoter activity in As4.1 cells, suggesting the pivotal role for the region in the regulation of the mouse renin gene. Electrophoretic mobility shift and supershift assays have identified two nuclear factor I-binding sites and a Sp1/Sp3-binding site within the distal portion of the region (−197 to −103). Mutation of these three sites caused a 90% decrease in Ren-1 c promoter activity. Mutational analysis and electrophoretic mobility shift assays have also identified three additional transcription factor-binding sites within the region from −103 to −69, each of which contributes to high-level expression of Ren-1 c in As4.1 cells. Finally, we have shown that the Ren-1 c enhancer is the target for endothelin-1 (ET-1)-induced inhibition of Ren-1 c expression and the transcription factor-binding sites in the proximal promoter are required for the maximal ET-1 inhibitory effect.

scholarly journals Regulation of chicken vanin1 gene expression by peroxisome proliferators activated receptor α and miRNA-181a-5p

2021 ◽  
Vol 34 (2) ◽  
pp. 172-184
Author(s):  
Zhongliang Wang ◽  
Jianfeng Yu ◽  
Nan Hua ◽  
Jie Li ◽  
Lu Xu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Binding Sites ◽  
Promoter Region ◽  
Transcription Factor Binding Sites ◽  
Transcription Factor Binding ◽  
Expression Level ◽  
Luciferase Reporter ◽  
Peroxisome Proliferators ◽  
Factor Binding

Objective: Vanin1 (VNN1) is a pantetheinase that can catalyze the hydrolysis of pantetheine to produce pantothenic acid and cysteamine. Our previous studies showed that <i>VNN1</i> is specifically expressed in chicken liver. In this study, we aimed to investigate the roles of peroxisome proliferators activated receptor α (PPARα) and miRNA-181a-5p in regulating <i>VNN1</i> gene expression in chicken liver.Methods: 5′-RACE was performed to identify the transcription start site of chicken <i>VNN1</i>. JASPAR and TFSEARCH were used to analyze the potential transcription factor binding sites in the promoter region of chicken <i>VNN1</i> and miRanda was used to search miRNA binding sites in 3′ untranslated region (3′UTR) of chicken <i>VNN1</i>. We used a knock-down strategy to manipulate PPARα (or miRNA-181a-5p) expression levels <i>in vitro</i> to further investigate its effect on <i>VNN1</i> gene transcription. Luciferase reporter assays were used to explore the specific regions of VNN1 targeted by PPARα and miRNA-181a-5p.Results: Sequence analysis of the VNN1 promoter region revealed several transcription factor-binding sites, including hepatocyte nuclear factor 1α (HNF1α), PPARα, and CCAAT/enhancer binding protein α. GW7647 (a specific agonist of PPARα) increased the expression level of <i>VNN1</i> mRNA in chicken primary hepatocytes, whereas knockdown of PPARα with siRNA increased VNN1 mRNA expression. Moreover, the predicted PPARα-binding site was confirmed to be necessary for PPARα regulation of <i>VNN1</i> gene expression. In addition, the <i>VNN1</i> 3′UTR contains a sequence that is completely complementary to nucleotides 1 to 7 of miRNA-181a-5p. Overexpression of miR-181a-5p significantly decreased the expression level of <i>VNN1</i> mRNA.Conclusion: This study demonstrates that PPARα is an important transcriptional activator of <i>VNN1</i> gene expression and that miRNA-181a-5p acts as a negative regulator of <i>VNN1</i> expression in chicken hepatocytes.

Sign in / Sign up

Export Citation Format

Share Document