Department of Physics, Massachusetts Institute of Technology, Cambridge, MA 02139.
Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA 02139.
Proc Natl Acad Sci U S A. 2023 Mar 14;120(11):e2210480120. doi: 10.1073/pnas.2210480120. Epub 2023 Mar 10.
Cohesin folds mammalian interphase chromosomes by extruding the chromatin fiber into numerous loops. "Loop extrusion" can be impeded by chromatin-bound factors, such as CTCF, which generates characteristic and functional chromatin organization patterns. It has been proposed that transcription relocalizes or interferes with cohesin and that active promoters are cohesin loading sites. However, the effects of transcription on cohesin have not been reconciled with observations of active extrusion by cohesin. To determine how transcription modulates extrusion, we studied mouse cells in which we could alter cohesin abundance, dynamics, and localization by genetic "knockouts" of the cohesin regulators CTCF and Wapl. Through Hi-C experiments, we discovered intricate, cohesin-dependent contact patterns near active genes. Chromatin organization around active genes exhibited hallmarks of interactions between transcribing RNA polymerases (RNAPs) and extruding cohesins. These observations could be reproduced by polymer simulations in which RNAPs were moving barriers to extrusion that obstructed, slowed, and pushed cohesins. The simulations predicted that preferential loading of cohesin at promoters is inconsistent with our experimental data. Additional ChIP-seq experiments showed that the putative cohesin loader Nipbl is not predominantly enriched at promoters. Therefore, we propose that cohesin is not preferentially loaded at promoters and that the barrier function of RNAP accounts for cohesin accumulation at active promoters. Altogether, we find that RNAP is an extrusion barrier that is not stationary, but rather, translocates and relocalizes cohesin. Loop extrusion and transcription might interact to dynamically generate and maintain gene interactions with regulatory elements and shape functional genomic organization.
黏合蛋白通过将染色质纤维挤出成许多环来折叠哺乳动物间期染色体。“环挤出”可能会受到染色质结合因子的阻碍，如 CTCF，它会产生特征性和功能性的染色质组织模式。有人提出转录会使黏合蛋白重新定位或干扰其功能，并且活跃的启动子是黏合蛋白加载的位点。然而，转录对黏合蛋白的影响与黏合蛋白的活性挤出观察结果尚未得到协调。为了确定转录如何调节挤出，我们研究了可以通过遗传“敲除”黏合蛋白调节剂 CTCF 和 Wapl 来改变黏合蛋白丰度、动力学和定位的小鼠细胞。通过 Hi-C 实验，我们发现了活跃基因附近复杂的、依赖黏合蛋白的接触模式。活跃基因周围的染色质组织表现出转录 RNA 聚合酶（RNAP）和挤出黏合蛋白之间相互作用的特征。这些观察结果可以通过聚合模拟来重现，其中 RNAP 是挤出的移动障碍，会阻碍、减缓和推动黏合蛋白。模拟预测，优先在启动子加载黏合蛋白与我们的实验数据不一致。额外的 ChIP-seq 实验表明，假定的黏合蛋白加载器 Nipbl 并非主要富集在启动子上。因此，我们提出黏合蛋白不是优先加载在启动子上，而是 RNAP 的屏障功能导致了活跃启动子上黏合蛋白的积累。总之，我们发现 RNAP 是一种挤出障碍，它不是固定的，而是可以迁移和重新定位黏合蛋白。环挤出和转录可能相互作用，以动态生成和维持基因与调节元件的相互作用，并塑造功能基因组组织。
