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

Abstract

四股螺旋結構 (G-quadruplexs) 是由含有許多重複鳥糞嘌呤 (Guanine) 的DNA序列所形成， G-quadruplexs二級結構容易穩定的在端粒和一些啟動子區域的原致癌基因上形成，我們發現WNT1啟動子上帶有一段G-rich的序列，利用可穩定G-quadruplex結構的化合物BMVC和BMVC4，這兩種G-quadruplex stabilizers藥物可以幫助WNT1啟動子上的G-quadruplex形成，並抑制下游WNT1基因的表現，為了進一步了解是否抑制的現象是經由G-quadruplex的結構，我們利用點突變的序列做in vitro (CD and NMR)的分析以及螢光reporter的分析，來看wild-type和mutants的WNT1啟動子活性，從實驗結果中，的確G-quadruplex stabilizers可以抑制wild-type WNT1 promoter的活性，且顯示是屬於dose-dependent的方式，而mutants則不受藥物影響是因為不會形成G-quadruplex結構，除此之外，為了了解是否藥物影響Wnt1蛋白的表現是因改變蛋白質的結合，在ChIP分析中，Nucleolin結合到G-quadruplex的量有增加，顯示WNT1啟動子在藥物的處理下會形成穩定的G-quadruplex結構，而抑制Wnt1蛋白的表現。另外我們想知道是否藥物可以可以影響到其他啟動子G-quadruplex的形成，例如BMP2和EGFR啟動子，從微陣列基因分析BMP2的表現會被抑制，因此我們想知道透過G-quadruplex stabilizers抑制Bmp2是否可使EMT過程轉變，實驗指出Bmp2及EMT現象不受到G-quadruplex stabilizers影響，由於藥物無法作用到BMP2啟動子而使G-quadruplex結構無法形成；EGFR啟動子帶有兩段GFS，亦有機會形成G-quadruplex，因此我們利用螢光reporter分析GFS的功能為何，以分析是否藥物可以影響到EGFR的表現，在NMR氫圖譜證明-137 bps和-376 bps的GFS在in vitro下有形成G-quadruplex的訊號，從repoter分析結果中，藥物處理下可增加-137 bps GFS的EGFR啟動子之活性。

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.