Analysis of the mechanisms controlling hDlg gene expression.

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

Mar del Plata

Congreso; XLIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2007

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB)

There is a great deal of evidence suggesting that hDlg expression is lost during tumour development in different tissues, associated with lack of cell polarity and tissue architecture. However, the mechanisms controlling hDlg expression are not fully understood. We performed the cloning and analysis of the genomic 5´flanking region of hDlg ORF with promoter activity, and determined minimal and cis elements required for efficient transcription. Using RACE techniques, we identified an alternative splicing event in the 5’ UTR of hDlg mRNA. We showed by reporter assays that the Dlg 5’-UTR containing the alternatively-spliced exon interferes with the translation of a downstream ORF, suggesting that this splicing event can contribute to regulate hDlg levels. Moreover, we found within hDlg promoter consensus binding sites for the Snail family of transcription factors that repress the expression of epithelial markers and are up-regulated in tumours. By co-transfection experiments we demonstrated that Snail proteins repressed transcriptional activity of hDlg promoter constructs and, in addition, stable expression of Snail in Caco2 cells leads to a consistently reduction of hDlg protein levels. To assess the direct interaction of Snail proteins with hDlg promoter sequences at the chromatin level, Chromatin immunoprecipitation (CHIP) assays are currently being performed.