Dépôt numérique

Transcriptional activity of the rat claudin-1 5 ' untranslated region in epididymal cells: Identification of a SP1 binding site

Dufresne, Julie et Cyr, Daniel G. ORCID logoORCID: https://orcid.org/0000-0002-6566-783X (2006). Transcriptional activity of the rat claudin-1 5 ' untranslated region in epididymal cells: Identification of a SP1 binding site In: 31st Annual Meeting of the American Society of Andrology, April 08-11, 2006, Chicago, IL.

Ce document n'est pas hébergé sur EspaceINRS.


Claudin-1 (Cldn-1) is a member of the claudin family of transmembrane proteins that are implicated in the formation of tight junctions. Cldn-1 has been shown to be present in epididymal tight junctions which are responsible for the formation of the blood-epididymal barrier. Studies from our laboratory have shown that in the initial segment of the epididymis Cldn-1 is an androgen regulated protein. There is little information on the regulation of the Cldn-1 promoter. In human epithelial cells it has been reported that the expression of the Cldn-1 gene is inhibited by two transcription factors: Snail and Slug. There is limited information on the factors that stimulate Cldn-1 expression. In the epididymis, there is no information on the regulation of 5' flanking region of genes coding for tight junctional proteins. Clearly the understanding of how genes such as Cldn-1 are regulated may provide essential information for understanding how the formation of the bloodepididymal barrier is regulated. The objective of the present study was to investigate the transcriptional regulation of the Cldn-1 gene in the rat epididymis. To address this, we first used a commercial genome walking kit and two gene-specific reverse primers to amplify and clone 1 775 pb upstream of the translation start codon of the rat Cldn-1 gene. This sequence showed 96% homology with the Rattus norvegicus chromosome 11 genomic contig. The precise transcriptional start site was then assessed experimentally by Rapid Amplification of cDNA Ends (5' RACE) using the same gene-specific primers. Results indicated that we had isolated the complete 5' end of transcript encoding Cldn-1 and that this transcriptional start site, an adenosine located at the -1 98 position relative to the first codon and 26 pb upstream of the putative TATA box, is the only start site in the rat epididymis. The Cldn-1 promoter was inserted into a promoterless pGL3-Basic vector containing a firefly luciferase gene. The vector was subsequently used to transfect the rat ca put epididymal cell line (RCE-1 ). Transfected cells were maintained in culture for 24hrs, lysed and luciferase activity assessed. Results indicated that the promoter contained sufficient information to drive the expression of epididymal Cldn-1 . To determine the regulation of the rat epididymal Cldn-1 promoter, PCR was used to amplify the Nhe/!Kpnl fragment of DNA sequences of the Cldn-1 promoter region (-1 414, -61 3, -226, -1 73, -125, -61 and -7 to +164 relatively to the transcription start site) and PCR products were inserted in the Nhel!Kpn/ double digested plasmid, upstream of the firefly luciferase gene. Cells were transfected with both the Cldn-1 luciferase constructs and a phRLTK vector expressing the Reni//a luciferase to normalize for the transfection efficiency. The minimal promoter activity was achieved with the -61 construct which contains a TATA box as well as two potential consensus SP1 binding sites. To determine whether or not the SP1 consensus sequences were functional, electrophoretic mobility shift and supershift assays were done to confirm that the SP1 transcription factor was present in RCE-1 nuclear extracts and that it could bind to the Cldn-1 promoter. Nuclear extracts from the initial segment and caput epididymidis of adult Sprague-Dawley rats indicated that SP1 was present in the epididymis and that it also bound to this DNA element of the Cldn-1 promoter. Binding of the SP1 transcription factor appeared to be specific and could be out competed by an excess of unlabelled oligonucleotide but not by an excess of mutated unlabelled oligonucleotide. Results from this study indicate that SP1 binds to the Cldn-1 promoter region and that this interaction appears to be implicated in regulating the expression of Cldn-1 in the rat epididymis.

Type de document: Document issu d'une conférence ou d'un atelier
Informations complémentaires: Affiche scientifique 36
Mots-clés libres: -
Centre: Centre INRS-Institut Armand Frappier
Date de dépôt: 01 avr. 2024 19:32
Dernière modification: 01 avr. 2024 19:32
URI: https://espace.inrs.ca/id/eprint/14274

Gestion Actions (Identification requise)

Modifier la notice Modifier la notice