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Regulation of the CLDN3 gene in ovarian cancer cells
Hiroshi Honda1, Michael J Pazin, Theresa D'Souza
1Laboratory of Cellular and Molecular Biology, National Institute on Aging, Baltimore, Maryland, USA.
Abstract:
The claudin (CLDN) genes encode a family of proteins involved in the formation and function of tight junctions. CLDN gene expression is frequently altered in several human cancers, and in particular, CLDN3 and CLDN4 are commonly overexpressed in ovarian cancer. However, the mechanisms leading to the deregulation of these genes in cancer remain unclear. In the present study, we have examined the CLDN3 promoter and have identified a minimal region containing an Sp1 site crucial for its activity. In addition, we find that the CLDN3 promoter is regulated through epigenetic processes. Cells that express high levels of CLDN3 exhibit low DNA methylation and high histone H3 acetylation of the critical CLDN3 promoter region, and the reverse is observed in cells that do not express this gene. CLDN3-negative cells can be induced to express CLDN3 through treatment with DNA methyltransferase or histone deacetylase inhibitors. Interestingly, in vitro binding experiments, as well as chip assays show that Sp1 binds the unmethylated promoter much more efficiently, providing a mechanism for CLDN3 silencing in non-expressing cells. Finally, siRNA-mediated knockdown of Sp1 led to a significant decrease of CLDN3 expression at both the mRNA and protein levels, demonstrating a crucial role for this transcription factor in the regulation of CLDN3. Our data provide a basis for CLDN3 expression in ovarian cancer cells, as well as a mechanism for the silencing of this promoter in tumors lacking expression of claudin-3.
Insights
Claudin (CLDN) gene expression in ovarian cancer is regulated by epigenetic mechanisms. Sp1 transcription factor binds to the CLDN3 promoter, influencing gene silencing in non-expressing cells.
Area of Science:
- Molecular Biology
- Cancer Biology
- Epigenetics
Background:
- Claudin (CLDN) genes are crucial for tight junction formation and function.
- CLDN3 and CLDN4 are frequently overexpressed in ovarian cancer, but the regulatory mechanisms are unclear.
Purpose of the Study:
- To investigate the regulatory mechanisms of CLDN3 gene expression in ovarian cancer.
- To identify key factors and epigenetic modifications involved in CLDN3 deregulation.
Main Methods:
- Promoter analysis to identify critical regulatory regions.
- Epigenetic analysis including DNA methylation and histone acetylation.
- In vitro binding assays and ChIP assays to study Sp1 binding.
- siRNA-mediated knockdown of Sp1 to assess its role.
Main Results:
- A minimal region with an Sp1 binding site was identified as crucial for CLDN3 promoter activity.
- CLDN3 expression correlates with low DNA methylation and high histone acetylation at the promoter.
- Inhibitors of DNA methyltransferase and histone deacetylase induced CLDN3 expression in negative cells.
- Sp1 binds more efficiently to unmethylated promoters, and Sp1 knockdown significantly reduced CLDN3 expression.
Conclusions:
- Epigenetic modifications, specifically DNA methylation and histone acetylation, play a critical role in regulating CLDN3 expression.
- Sp1 is a key transcription factor that binds to the CLDN3 promoter and mediates its expression.
- These findings provide a mechanism for CLDN3 silencing in ovarian tumors lacking expression.
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