Marsden, Mark D.; (1995) A molecular genetic analysis of the desmosomal cadherins. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The desmocollins and the desmogleins belong to the cadherin superfamily of adhesion molecules and are present in the desmosome, a specialised intercellular junction characteristic of epithelium. Three types of desmocollin and three types of desmoglein have been identified and in desmosome-bearing tissues such as epidermis these are differentially expressed. Both DSC1 and DSC2 encode desmocollin isoforms. Whilst the expression of DSC1 is predominantly suprabasal in epidermis, DSC2 is expressed in all desmosome-bearing tissues. To initiate an investigation into the mechanisms that regulate the differential expression of these genes, human genomic libraries were screened to isolate 5' DNA regulatory elements. λ clones containing the DSC2 promoter were identified by screening a human genomic library by hybridisation to the most 5' 197bp of the human DSC2 cDNA. Analysis of such a 5' genomic clone by RNase protection experiments revealed a major transcription initiation site 201bp upstream of the translation start site. Analysis of the 1.9kb DNA sequence upstream of the DSC2 translation start site revealed consensus binding sites for transcription factors associated with the epidermis and motifs common to the promoters of other epidermally expressed genes. Southern analysis confirmed the presence of a CpG island by the presence of clustered restriction sites for the methylation-sensitive endonucleases BssHII and EagI. The activity of the human DSC2 promoter, was examined by coupling it to the luciferase reporter gene. Transient transfection of this reporter construct and also deletions derived from it, into epithelial and non-epithelial cell lines enabled activating and inhibitory DNA elements to be mapped, and identified cell-specific elements between -332 and -525 (relative to translation start site at 0). Other work undertaken as preliminary to gene knockout experiments has been the isolation of a mouse genomic clone for DSC2, and the sequence analysis of a previously isolated mouse desmoglein cDNA.

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