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First published online 28 September 2004
doi: 10.1242/jcs.01399

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Disruption of the cingulin gene does not prevent tight junction formation but alters gene expression

¹ Department of Molecular Biology, University of Geneva, 30 Quai Ernest-Ansermet, 1211 Genève 4, Switzerland
² NCCR `Frontiers in Genetics', University of Geneva, 30 Quai Ernest-Ansermet, 1211 Genève 4, Switzerland
³ Department of Cell Physiology and Metabolism, CMU, University of Geneva, 1 Rue Michel Servet, 1211 Genève 4, Switzerland
⁴ Department of Biology, University of Padova, Via U. Bassi 58B, 35121 Padova, Italy

^* Author for correspondence (e-mail: sandra.citi{at}molbio.unige.ch)

Accepted 6 July 2004

Cingulin, a component of vertebrate tight junctions, contains a head domain that controls its junctional recruitment and protein interactions. To determine whether lack of junctional cingulin affects tight-junction organization and function, we examined the phenotype of embryoid bodies derived from embryonic stem cells carrying one or two alleles of cingulin with a targeted deletion of the exon coding for most of the predicted head domain. In homozygous (–/–) embryoid bodies, no full-length cingulin was detected by immunoblotting and no junctional labeling was detected by immunofluorescence. In hetero- and homozygous (+/– and –/–) embryoid bodies, immunoblotting revealed a Triton-soluble, truncated form of cingulin, increased levels of the tight junction proteins ZO-2, occludin, claudin-6 and Lfc, and decreased levels of ZO-1. The +/– and –/– embryoid bodies contained epithelial cells with normal tight junctions, as determined by freeze-fracture and transmission electron microscopy, and a biotin permeability assay. The localization of ZO-1, occludin and claudin-6 appeared normal in mutant epithelial cells, indicating that cingulin is not required for their junctional recruitment. Real-time quantitative reverse-transcription PCR (real-time qRT-PCR) showed that differentiation of embryonic stem cells into embryoid bodies was associated with up-regulation of mRNAs for several tight junction proteins. Microarray analysis and real-time qRT-PCR showed that cingulin mutation caused a further increase in the transcript levels of occludin, claudin-2, claudin-6 and claudin-7, which were probably due to an increase in expression of GATA-6, GATA-4 and HNF-4, transcription factors implicated in endodermal differentiation. Thus, lack of junctional cingulin does not prevent tight-junction formation, but gene expression and tight junction protein levels are altered by the cingulin mutation.

Key words: Cingulin, Tight junction, Epithelium, Differentiation, Embryoid body, ES cell

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