Department of Chemistry, Lomonosov Moscow State University, Leninskie Gory 1, 119991 Moscow, Russia.
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskie Gory 1, 119991 Moscow, Russia.
Int J Mol Sci. 2023 Dec 19;25(1):45. doi: 10.3390/ijms25010045.
The methylation of cytosines at CpG sites in DNA, carried out de novo by DNA methyltransferase Dnmt3a, is a basic epigenetic modification involved in gene regulation and genome stability. Aberrant CpG methylation in gene promoters leads to oncogenesis. In oncogene promoters, CpG sites often colocalize with guanine-rich sequences capable of folding into G-quadruplexes (G4s). Our in vitro study aimed to investigate how parallel G4s formed by a sequence derived from the oncogene promoter region affect the activity of the Dnmt3a catalytic domain (Dnmt3a-CD). For this purpose, we designed synthetic oligonucleotide constructs: a G4-forming oligonucleotide and linear double-stranded DNA containing an embedded stable extrahelical G4. The topology and thermal stability of G4 structures in these DNA models were analyzed using physicochemical techniques. We showed that Dnmt3a-CD specifically binds to an oligonucleotide containing G4, resulting in inhibition of its methylation activity. G4 formation in a double-stranded context significantly reduces Dnmt3a-CD-induced methylation of a CpG site located in close proximity to the quadruplex structure; this effect depends on the distance between the non-canonical structure and the specific CpG site. One would expect DNA hypomethylation near the G4 structure, while regions distant from this non-canonical form would maintain a regular pattern of high methylation levels. We hypothesize that the G4 structure sequesters the Dnmt3a-CD and impedes its proper binding to B-DNA, resulting in hypomethylation and activation of transcription.
DNA 中 CpG 位点的胞嘧啶甲基化是一种基本的表观遗传修饰,涉及基因调控和基因组稳定性。异常的 CpG 甲基化会导致癌基因的发生。在癌基因启动子中,CpG 位点通常与富含鸟嘌呤的序列聚集在一起,这些序列能够折叠成 G-四链体 (G4)。我们的体外研究旨在研究源自癌基因启动子区域的序列形成的平行 G4 如何影响 Dnmt3a 催化结构域 (Dnmt3a-CD) 的活性。为此,我们设计了合成寡核苷酸构建体:一个形成 G4 的寡核苷酸和一个包含嵌入稳定的非螺旋 G4 的线性双链 DNA。使用物理化学技术分析了这些 DNA 模型中 G4 结构的拓扑结构和热稳定性。我们表明,Dnmt3a-CD 特异性结合含有 G4 的寡核苷酸,导致其甲基化活性受到抑制。在双链背景下形成 G4 会显著降低 Dnmt3a-CD 诱导的紧邻四链体结构的 CpG 位点的甲基化;这种效应取决于非规范结构与特定 CpG 位点之间的距离。人们预计在 G4 结构附近的 DNA 会出现低甲基化,而远离这种非规范形式的区域则会保持高甲基化水平的正常模式。我们假设 G4 结构将 Dnmt3a-CD 隔离,并阻碍其与 B-DNA 的正确结合,从而导致低甲基化和转录激活。