Cloning and functional characterization of Dlg tumour suppressor promoter

CAVATORTA, ANA L.; ADRIANA ANGELICA GIRI; BANKS, LAWRENCE; GARDIOL, DANIELA

Rosario

Congreso; XLII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2006

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Human Discs large (Dlg) was shown to be targeted by HPV E6 oncoproteins and to be down-regulated in HPV-associated cervical carcinomas. The knowledge about Dlg oncosuppressor functions has progressed in  the last years, and it was 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. Therefore, we proceeded to clone and characterise the Dlg promoter and analyse its transcriptional regulation. We cloned a 5´flanking region of Dlg ORF that exhibited promoter functions in different cell lines. We further analyzed the activity of a series of 5’ deletion constructs of the Dlg promoter and we could determine the minimal essential sequences that are required for promoter activity and domains that activate or inhibit transcription. Using bioinformatics tools, we found binding sites for the Snail family of transcription factors that repress other oncosuppressors and are up-regulated in tumours. By co-transfection experiments and luciferase assays we could demonstrate that Snail proteins repress also Dlg promoter and, in addition, mutations within the consensus sites that bind Snail, consistently diminished the inhibitory effect. The significance and involvement of Snail proteins in regulating endogenous Dlg levels are currently being analyzed.