Functional impact of regulatory polymorphisms (rSNP) in G1/S cell cycle checkpoint genes

¹Joëlle Dionne, ¹Manon Ouimet, ¹Vincent Gagné, ¹Mathieu Larivière, ^1,2Damian Labuda, ^1,2Daniel Sinnett
¹Division of Hematology-Oncology, research center, Ste. Justine Medical University Center, University of Montreal, Montreal, Canada, ²Department of Pediatrics, University of Montreal, Montreal, Canada

G1/S transition in the cell cycle is a finely regulated biological process. Depending on the context, growth-control genes present in the G1/S checkpoint can stop the cell cycle progression and activate survival pathway in the cell. Then DNA repair process or cell death by apoptosis is initiated. Dysregulation of G1/S checkpoint genes is frequently observed in complex disease, particularly like cancer. Indeed gene encoding components of cell cycle processes are frequently mutated in human cancers. We hypothesize that functional polymorphisms located in the regulatory region of candidates genes could lead to variable level of transcript and thus predisposing the individuals carrying these genetic variants to cancer. In this report we assessed the functional impact of rSNPs located in the proximal promoter of 18 candidates genes encoding components of G1/S checkpoint by combining in silico analysis and in vitro functional assays. We identified 150 rSNPs including 123 with predicted impact on putative transcription factor binding sites. This information was used to construct promoter haplotypes (rHAP). Following the subcloning of the major promoter haplotypes into a luciferase gene reporter vector (pGL3b), transient transfection assays were performed in 3 cell lines (Hela, Jeg3 and HepG2). We found that at least 11 rHAPs associated with TGFB1, TFDP1, CDKN1A, CDKN1B, CDKN2A and CDKN2B significantly influenced transcriptional activity in an allele-specific manner. Further validation by electrophoresis mobility shift assays (EMSA) to detect differential DNA-protein bindings is being done. Although, the biological significance of these observations still remain to be demonstrated, the expected variability of expression levels in key cell cycle components might influence individual's risk of cancer.