Homotypic chromatin interactions and loop extrusion are thought to be the two main drivers of mammalian chromosome folding. Here we tested the role of RNA polymerase II (RNAPII) across different scales of interphase chromatin organization in a cellular system allowing for its rapid, auxin-mediated degradation. We combined Micro-C and computational modeling to characterize subsets of loops differentially gained or lost upon RNAPII depletion. Gained loops, extrusion of which was antagonized by RNAPII, almost invariably formed by engaging new or rewired CTCF anchors. Lost loops selectively affected contacts between enhancers and promoters anchored by RNAPII, explaining the repression of most genes. Surprisingly, promoter–promoter interactions remained essentially unaffected by polymerase depletion, and cohesin occupancy was sustained. Together, our findings reconcile the role of RNAPII in transcription with its direct involvement in setting-up regulatory three-dimensional chromatin contacts genome wide, while also revealing an impact on cohesin loop extrusion.

同型染色质相互作用和环挤压被认为是哺乳动物染色体折叠的两个主要驱动因素。在这里，我们测试了 RNA 聚合酶 II (RNAPII) 在细胞系统中不同尺度的间期染色质组织中的作用，允许其快速、生长素介导的降解。我们结合 Micro-C 和计算模型来表征 RNAPII 耗尽时差异获得或丢失的循环子集。获得的环，其挤出受到 RNAPII 的拮抗，几乎总是通过接合新的或重新连接的 CTCF 锚而形成。丢失的环选择性地影响由 RNAPII 锚定的增强子和启动子之间的接触，解释了大多数基因的抑制。令人惊讶的是，启动子-启动子之间的相互作用基本上不受聚合酶消耗的影响，并且粘连蛋白占据保持不变。总之，我们的研究结果协调了 RNAPII 在转录中的作用及其直接参与建立全基因组调节性三维染色质接触的过程，同时还揭示了对粘连蛋白环挤出的影响。