分析WNT-1, BMP2及EGFR啟動子之G-quadruplex形成序列

Abstract

DNA sequences with tandem guanine repeats tend to from G-quadruplex (G4) structure. G-quadruplex is easily formed in telomere and the promoter regions of many proto-oncogenes. Previously our lab has found a G-rich sequences located at the WNT1 promoter is capable of forming G-quadruplex. The expression of Wnt1 was repressed by G-quadruplex stabilizers. To determine whether the repression is G-quadruplex structure dependent, we first analyzed mutations on these G-rich sequences using CD and NMR. Mutants that failed to form G-quadruplex were identified. I then established a luciferase reporter assay to monitor the WNT1 promoter expression. Our results revealed that wild-type WNT1 promoter can be suppressed by G-quadruplex stabilizers in a dose-dependent manner. Mutants that failed to form G-quadruplex structure were not repressed by drugs. The binding of nucleolin is also tested to check whether the repression of expression by BMVC is due to a change in protein binding. Using ChIP assay, we found BMVC increased the binding of nucleolin to WNT1 promoter, suggesting that the G-quadruplex structure was formed at the WNT1 promoter upon drug treatment. Thus, our results indicated that formation of the G-quadruplex structure at WNT1 promoter repressed its expression. We also identified G-qaudruplex-forming sequences on BMP2 and EGFR promoters. From microarray results, we found BMP2 is repressed by G-quadruplex stabilizers. Our results showed Bmp2 is not repressed due to G-quadruplex structure cannot be formed in cell level, suggesting our drugs cannot target to BMP2 promoter. We determine two G-qaudruplex-forming sequences on EGFR promoter functional roles by luciferase reporter assay and the ability to form G-quadruplex in vitro. And it can induce -137 GFS EGFR promoter activity in G-quadruplex stabilizers treament.