Molecular cloning of a functional promoter of the human plakoglobin gene

Objective: Plakoglobin (Pg) is the only cytoplasmic protein component commo n to both junctional complexes mediating cell-cell adhesion, adherens junct ions and desmosomes. In these complexes Pg appears to act as a linker prote in anchoring transmembrane proteins of the cadherin superfamily to the acti n cytoskeleton and intermediate filament system respectively. Intercellular adhesion is frequently disturbed in skin diseases and in carcinomas, enabl ing tumour progression and metastasis. Whereas Pg expression is lost in som e thyroid tumours and carcinoma cell lines, little information on Pg gene r egulation is currently available owing to a lack of promoter studies. Design and methods: We have cloned and sequenced genomic DNA from a human l ibrary that resulted in 979 bp upstream of the published Pg cDNA. The trans criptional start was mapped by rapid amplification of cDNA ends. Methylatio n-specific PCR of bisulfite-modified cell fine DNA was applied to probe the methylation status of a promoter-associated CpG island. Reporter-gene cons tructs of various promoter fragments were transiently transfected in thyroi d carcinoma cell lines and their activities were determined by luciferase m easurements. Results and conclusions: A I kb DNA fragment harbouring a functional promot er of the human Pg gene was cloned and characterized. The sequence lacks a canonical TATA box, but contains putative CCAAT boxes as well as various pu tative binding sites for transcription factors, among them SP I and AP2, pr oximal to the transcriptional start. Considerable promoter activity was fou nd in thyroid cell lines and deletion analysis indicated that a 300 bp regi on proximal to the 5'-untranslated region of the mRNA represents the minima l promoter of the human Pg gene. As cells lacking endogenous Pg expression were found to contain methylated CpG dinucleotides in a CpG island located around the transcriptional start site, it is suggested that epigenetic mech anisms such as DNA methylation contribute to dysregulated Pg expression.