High-precision mapping of nuclear pore-chromatin interactions reveals new principles of genome organization at the nuclear envelope. 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.

核孔复合物（nuclear pore complexes, NPCs）在基因组组织中的调控作用仍未被充分阐释，这是由于目前尚无法特异性探测核膜相关的全基因组蛋白质-DNA相互作用。为此，我们开发了一种高灵敏度的方法——NPC-DamID，该方法将核输入过程的体外重建体系与DamID技术相结合。这种无需固定样品的方法可特异性识别完整细胞核中核孔复合物结合的基因组DNA相互作用位点。我们发现，在多种细胞及组织类型中，核孔复合物优先与保守且具有层级排布的超级增强子（super-enhancers, SEs）相结合。我们还在核孔复合物处发现了相分离凝聚体，这类凝聚体可在超级增强子调控的基因位点实现区室化，并富集转录共激活因子与结构蛋白。我们的研究结果支持如下假说：核孔复合物可通过与CCCTC结合因子（CTCF）相互作用，作为锚定超级增强子调控枢纽的位点；同时还可通过与Med1、RNA聚合酶II（Pol II）等超级增强子结构蛋白的相互作用，维持超级增强子的三维构象。

实验整体设计：本研究采用名为NPC-DamID的全新方法开展测序实验。简言之，将融合了输入蛋白β（Importin β）的Dam蛋白与其他可在核孔复合物（NPC）附近实现DNA甲基化的试剂共同添加至半透化细胞中。实验结束后分离基因组DNA，先用DpnI限制性内切酶（DpnI）进行酶切，随后进行接头连接，并对DpnI酶切得到的片段进行PCR扩增。该流程与传统DamID实验流程高度相似。扩增产物经PCR纯化试剂盒纯化后，进行超声破碎，再用AlwI限制性内切酶（AlwI）酶切以去除接头序列。之后再次通过PCR纯化试剂盒纯化片段，最终使用Hyper-Kapa试剂盒（Hyper-Kapa）构建测序文库。