G-quadruplexes associated with R-loops promote CTCF binding. Wulfridge et al.

CTCF is a critical regulator of genome architecture and gene expression that binds to thousands of sites on chromatin. CTCF genomic localization is controlled by its interaction to a DNA sequence motif and regulated by DNA modifications. However, CTCF does not bind to all its potential sites in all cell types, raising the question of whether the underlying chromatin structure can regulate CTCF binding. Here we report that R-loops, three-stranded chromatin structures that contain DNA:RNA hybrids, facilitate CTCF binding through formation of the associated G-quadruplex (G4) secondary DNA structure. R-loops and G4 structures are co-localized with CTCF at many genomic regions in vivo and promote CTCF binding to its cognate DNA motif in vitro. Removal of R-loops and local deletion of a G4 forming motif reduce CTCF binding, which in turn can alter gene expression. Conversely, chemical stabilization of G4s in undifferentiated stem cells results in CTCF gains and accompanying alterations in chromatin organization. Finally, G4 induction upon neurodifferentiation is associated with CTCF increase at bivalent genes. Our results suggest that formation of G4 structures by R-loops plays a pivotal role in reinforcing long range chromatin interactions through CTCF.CTCF（CCCTC结合因子）是基因组架构与基因表达的关键调控因子，可结合染色质上的数千个位点。CTCF的基因组定位受其与DNA序列基序的相互作用调控，并受DNA修饰的调节。然而，CTCF并非在所有细胞类型中都结合其所有潜在结合位点，这引发了一个核心科学问题：染色质的固有结构是否能够调控CTCF的结合行为。本研究报道，R环（R-loops）——一种包含DNA:RNA杂交链的三链染色质结构——可通过形成与之相关的G-四链体（G-quadruplex，G4）DNA二级结构，促进CTCF的结合。R环与G4结构在体内诸多基因组区域中与CTCF共定位，并在体外能够促进CTCF与其同源DNA基序的结合。去除R环以及局部缺失形成G4的基序会削弱CTCF的结合能力，进而改变基因表达模式。反之，在未分化干细胞中对G4进行化学稳定处理，可诱导CTCF结合增加，并伴随染色质组织的相应改变。最终，神经分化过程中G4的诱导与二价基因位点上CTCF结合的增强存在关联。我们的研究结果表明，由R环介导形成的G4结构在通过CTCF强化长距离染色质相互作用这一过程中发挥关键调控作用。