Analysis of the regulatory mechanisms that control Discs Large tumour suppresor gene expression

CAVATORTA, ANA LAURA; GIRI, ADRIANA A.; BANKS, LAWRENCE; GARDIOL, DANIELA

Praga, Republica Checa

Conferencia; 23rd International Papillomavirus Conference; 2006

Human Discs large (Dlg) was shown to be targeted by HPV E6 oncoproteins and to be down-regulated during malignant progression in HPV-associated cervical carcinomas. The knowledge about Dlg oncosuppressor functions in mammalian cells has progressed in the last years, and a large number of evidences demonstrated its involvement in both cell polarity control and tissue architecture maintenance. However, the mechanisms controlling Dlg expression at the transcriptional level are still unknown. This is particularly relevant since Dlg is lost in many tumours in both HPV E6 dependent and independent manners. Therefore, we proceeded to clone and characterise the Dlg promoter and analyse its transcriptional regulation. By bioinformatic analysis, a potential Dlg promoter region was identified in the 5´flanking region of Dlg ORF. Fragments of this region were amplified, cloned into a reporter vector and the promoter activity was determined in different cell lines by luciferase assays. We further analyzed the activity of a series of 5’ deletion constructs of the Dlg promoter region and we could determine the minimal essential sequences that are required for promoter activity as well as cis elements that activate or inhibit transcription. By RACE methodologies we investigated the start transcription sites of Dlg and we discovered the presence of an alternatively-spliced exon in the 5’-UTR of Dlg mRNA that may contribute to regulation of Dlg expression. Using bioinformatic tools, binding sites for different transcription factors were found in this region. The involvement of one tumour-related factor in Dlg regulation is currently being analysed.