GSE176106
High-precision mapping of nuclear pore-chromatin interactions reveals new principles of genome organization at the nuclear envelope
The role of nuclear pore complexes (NPCs) in genome organization remains poorly characterized due to technical limitations in probing genome-wide protein-DNA interactions that are specific to the nuclear envelope. Here, we developed a sensitive method, NPC-DamID, which combines in vitro reconstitution of nuclear import and DamID technology. This fixation-free method specifically identifies genomic DNA interactions at the NPCs in intact nuclei. We found that NPCs are preferentially associated with common and hierarchically arranged super-enhancers (SEs) across multiple cells and tissue types. We also uncovered phase-separated condensates at NPCs that compartmentalize and concentrate transcriptional coactivators and structural proteins at SE-regulated genes. Our results support the idea that NPCs are sites of anchoring SE regulatory hubs through their interaction with CTCF and also maintains the conformation of SEs through interaction with structural proteins of SEs like Med1 and PolII. Overall design: The sequencing experiemnts were done using a new method called NPC-DamID. Breifly, Dam fused to Importin beta was provided to semi-permeabilized cells along with other reagents for methylation of DNA in the vicinity of nuclear pore complexes (NPCs). The genomic DNA was isolated after the assay and digested with DpnI followed by adapter ligation and PCR amplification of the DpnI digested fragements. This is very similar to convention DamID pipeline. The amplified product was purified using PCR purification kit and then sonicated and digested with alwI for removing the adapters. After this the fragemnts were purified using PCR purification kit and sequencing libraries were prepared using Hyper-Kapa kit.