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.

CCCTC结合因子（CTCF）是基因组三维架构与基因表达的关键调控因子，可结合染色质上数千个靶位点。CTCF的基因组定位既依赖其与DNA序列基序的相互作用，又受DNA修饰的调控。然而，CTCF并不会在所有细胞类型中结合所有潜在结合位点，这引发了一个关键科学问题：染色质的固有结构是否能够调控CTCF的结合活性。本研究发现，一类包含DNA:RNA杂交链的三链染色质结构——R环（R-loops），可通过形成相关的G-四链体（G-quadruplex, G4）二级DNA结构，促进CTCF的结合。体内实验证实，R环与G4结构可在众多基因组区域与CTCF发生共定位；体外实验则表明，二者可促进CTCF与其同源DNA基序的结合。敲除R环或局部删除可形成G4的基序，会降低CTCF的结合活性，进而改变基因表达模式。反之，在未分化干细胞中通过化学手段稳定G4结构，可使CTCF结合位点增加，并伴随染色质组织的重塑。最后，神经分化过程中G4结构的诱导与二价基因位点上CTCF结合水平的升高显著相关。本研究结果表明，R环介导的G4结构形成，在通过CTCF强化远程染色质相互作用的过程中发挥关键调控作用。