Submitted September 14, 2006
Returned for revision October 20, 2006
Accepted January 17, 2007
Male Reproductive Tract
Activation of an SP Binding Site Is Crucial for the
Expression of Claudin 1 in Rat Epididymal Principal Cells
Julie Dufresne
and
Daniel G. Cyr *
* To whom correspondence should be addressed. E-mail: daniel.cyr{at}iaf.inrs.ca.
Abstract
Claudin 1 (CLDN1) is a tight junctional protein present in
the epididymis. Limited information exists regarding the
regulation of Cldn1 transcription. In the
epididymis, the regulation of the 5' flanking region of
genes coding for tight junctional proteins is unknown. The
present objectives were to investigate the transcriptional
regulation of the Cldn1 gene in the rat epididymis.
A 1.8kb sequence of the 5' flanking region of the rat
Cldn1 gene was cloned. The transcriptional start
site is an adenine located at the -198 position relative
to the first codon and 26 bp downstream of the putative
TATA box. It is the only start site for the Cldn1
gene transcription in the rat epididymis. The Cldn1
promoter was inserted into a luciferase gene expression
vector and transfected into a rat caput epididymal cell
line (RCE-1). Sequential deletion analysis revealed that
minimal promoter activity was achieved with the construct
containing -61 to +164 bp of the promoter. This sequence
contained a TATA box and two consensus SP1 binding sites.
Electrophoretic mobility shift and supershift assays
confirmed that SP1 and SP3 were present in RCE-1 cell and
epididymal nuclear extracts, and that they bind to the 5'
SP1 binding motif of the promoter. Site directed
mutagenesis of the 5' SP1 binding site resulted in a
4-fold decrease in transactivation of the minimal promoter
sequence. These findings indicate that SP1 binds to the
Cldn1 promoter region and that this interaction
influences the expression of Cldn1 in the rat
epididymis.
Key words:
Male Reproductive Tract •
Epididymis •
Gene regulation
