Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 119435 Moscow, Russia.
Moscow Institute of Physics and Technology, 141701 Dolgoprudny, Russia.
Int J Mol Sci. 2021 Jun 30;22(13):7090. doi: 10.3390/ijms22137090.
G-quadruplex (G4) sites in the human genome frequently colocalize with CCCTC-binding factor (CTCF)-bound sites in CpG islands (CGIs). We aimed to clarify the role of G4s in CTCF positioning. Molecular modeling data suggested direct interactions, so we performed in vitro binding assays with quadruplex-forming sequences from CGIs in the human genome. G4s bound CTCF with Kd values similar to that of the control duplex, while respective i-motifs exhibited no affinity for CTCF. Using ChIP-qPCR assays, we showed that G4-stabilizing ligands enhance CTCF occupancy at a G4-prone site in STAT3 gene. In view of the reportedly increased CTCF affinity for hypomethylated DNA, we next questioned whether G4s also facilitate CTCF recruitment to CGIs via protecting CpG sites from methylation. Bioinformatics analysis of previously published data argued against such a possibility. Finally, we questioned whether G4s facilitate CTCF recruitment by affecting chromatin structure. We showed that three architectural chromatin proteins of the high mobility group colocalize with G4s in the genome and recognize parallel-stranded or mixed-topology G4s in vitro. One of such proteins, HMGN3, contributes to the association between G4s and CTCF according to our bioinformatics analysis. These findings support both direct and indirect roles of G4s in CTCF recruitment.
人类基因组中的 G-四链体 (G4) 位点经常与 CpG 岛 (CGI) 中结合因子 (CTCF) 的结合位点共定位。我们旨在阐明 G4 在 CTCF 定位中的作用。分子建模数据表明存在直接相互作用，因此我们使用来自人类基因组 CGIs 的四链体形成序列进行了体外结合测定。G4 与 Kd 值与对照双链体相似的 CTCF 结合，而各自的 i- 基序则没有与 CTCF 的亲和力。通过 ChIP-qPCR 测定，我们表明 G4 稳定配体增强了 STAT3 基因中易于形成 G4 的位点处的 CTCF 占有率。鉴于据报道 CTCF 对低甲基化 DNA 的亲和力增加，我们接下来质疑 G4 是否也通过保护 CpG 位点免受甲基化来促进 CTCF 募集到 CGIs。对先前发表数据的生物信息学分析表明并非如此。最后，我们质疑 G4 是否通过影响染色质结构来促进 CTCF 的募集。我们表明，高迁移率族蛋白的三种结构染色质蛋白在基因组中与 G4 共定位，并在体外识别平行链或混合拓扑 G4。根据我们的生物信息学分析，此类蛋白质之一 HMGN3 有助于 G4 与 CTCF 之间的关联。这些发现支持 G4 在 CTCF 募集中的直接和间接作用。
