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Claudins form a large family of tight junction proteins that have essential roles in the control of paracellular ion flux and the maintenance of cell polarity. Many studies have shown that several claudin family members are abnormally expressed in various cancers. In particular, CLDN4 (encoding claudin-4) is overexpressed in ovarian cancer. However, although CLDN4 overexpression is well established, the mechanisms responsible for this abnormal regulation remain unknown. In the present study, we delineate a small region of the CLDN4 promoter critical for its expression. This region contains two Sp1 sites, both of which are required for promoter activity. However, because of the ubiquitous expression of Sp1, these sites, although necessary, are not sufficient to explain the patterns of gene expression of CLDN4 in various ovarian tissues. We show that the CLDN4 promoter is further controlled by epigenetic modifications of the Sp1-containing critical promoter region. Cells that overexpress CLDN4 exhibit low DNA methylation and high histone H3 acetylation of the critical CLDN4 promoter region, and the reverse is observed in cells that do not express CLDN4. Moreover, the CLDN4-negative cells can be induced to express CLDN4 through treatment with demethylating and/or acetylating agents. Because CLDN4 is elevated in a large fraction of ovarian cancer, the mechanism leading to deregulation may represent a general pathway in ovarian tumorigenesis and may lead to novel strategies for therapy and an overall better understanding of the biology of this disease.

Recently, the large filamentous striated-muscle protein titin has been observed in non-muscle cells, and, in one instance, has been proposed to have a nuclear function as a chromosomal component contributing to structure and elasticity. In this study, we sought to further characterize the presumptive nuclear isoform of titin. Immunofluorescence microscopy with multiple titin-specific monoclonal antibodies shows localization to the nucleus in interphase cells and to the spindle machinery in mitotic cells in all cell types examined; localization to condensed chromosomes is not observed. An abundant 700-kDa phosphoprotein is the predominant species immunoprecipitated with these antibodies. Sequencing of peptide fragments of the immunopurified protein reveals identity to AHNAK, a nuclear phosphoprotein, an identification that was confirmed by Western blot analysis with antibodies to AHNAK and peptide fragmentation patterns. Sequence comparison suggests similarities between the repetitive heptad phi+/-phiP+/-phi+/- motif in AHNAK and the PEVK region of titin, potentially explaining the cross-reactivity observed between AHNAK antibodies and titin antibodies. Interestingly, although some AHNAK antibodies stain interphase nuclei, no evidence of mitotic spindle localization is seen, suggesting that the identity of the protein at the latter location is more closely related to titin than AHNAK. This concept is further supported by observations that cell lines not expressing AHNAK have similar antititin antibody localization to the mitotic spindle. We conclude that (1) multiple titin antibodies, particularly those recognizing the PEVK region, cross-react with AHNAK, and (2) the mitotic spindle staining observed with antititin antibodies is most likely due to the association of titin or a titin-like molecule with this structure., (Copyright 2001 Wiley-Liss, Inc.)