Department of Medicine, University of Udine, 33100 Udine, Italy.
Nucleic Acid Center, Institute of Physics and Chemistry, University of Southern Denmark, DK-5230 Odense, Denmark.
Nucleic Acids Res. 2018 Jan 25;46(2):661-676. doi: 10.1093/nar/gkx1142.
KRAS is one of the most mutated genes in human cancer. It is controlled by a G4 motif located upstream of the transcription start site. In this paper, we demonstrate that 8-oxoguanine (8-oxoG), being more abundant in G4 than in non-G4 regions, is a new player in the regulation of this oncogene. We designed oligonucleotides mimicking the KRAS G4-motif and found that 8-oxoG impacts folding and stability of the G-quadruplex. Dimethylsulphate-footprinting showed that the G-run carrying 8-oxoG is excluded from the G-tetrads and replaced by a redundant G-run in the KRAS G4-motif. Chromatin immunoprecipitation revealed that the base-excision repair protein OGG1 is recruited to the KRAS promoter when the level of 8-oxoG in the G4 region is raised by H2O2. Polyacrylamide gel electrophoresis evidenced that OGG1 removes 8-oxoG from the G4-motif in duplex, but when folded it binds to the G-quadruplex in a non-productive way. We also found that 8-oxoG enhances the recruitment to the KRAS promoter of MAZ and hnRNP A1, two nuclear factors essential for transcription. All this suggests that 8-oxoG in the promoter G4 region could have an epigenetic potential for the control of gene expression.
KRAS 是人类癌症中突变最频繁的基因之一。它受转录起始位点上游的 G4 基序控制。在本文中，我们证明了 8-氧鸟嘌呤（8-oxoG）在 G4 中比在非 G4 区域更丰富，是调节这种致癌基因的新成员。我们设计了模拟 KRAS G4 基序的寡核苷酸，并发现 8-oxoG 影响 G-四链体的折叠和稳定性。二甲磺酸足迹显示，携带 8-oxoG 的 G-链被排除在 G-四联体之外，并用 KRAS G4 基序中的冗余 G-链取代。染色质免疫沉淀显示，当 G4 区域的 8-oxoG 水平因 H2O2 而升高时，碱基切除修复蛋白 OGG1 被募集到 KRAS 启动子。聚丙烯酰胺凝胶电泳证明 OGG1 可以从双链体中的 G4 基序中去除 8-oxoG，但当它折叠时，它以非生产性方式结合到 G-四链体上。我们还发现 8-oxoG 增强了 MAZ 和 hnRNP A1 两种核因子到 KRAS 启动子的募集，这两种核因子对转录至关重要。所有这些都表明启动子 G4 区域的 8-oxoG 可能具有控制基因表达的表观遗传潜力。
