Cohesin and CCCTC-binding factor (CTCF) are key regulatory proteins of three-dimensional (3D) genome organization. Cohesin extrudes DNA loops that are anchored by CTCF in a polar orientation. Here, we present direct evidence that CTCF binding polarity controls cohesin-mediated DNA looping. Using single-molecule imaging, we demonstrate that a critical N-terminal motif of CTCF blocks cohesin translocation and DNA looping. The cryo-EM structure of the cohesin-CTCF complex reveals that this CTCF motif ahead of zinc fingers can only reach its binding site on the STAG1 cohesin subunit when the N terminus of CTCF faces cohesin. Remarkably, a C-terminally oriented CTCF accelerates DNA compaction by cohesin. DNA-bound Cas9 and Cas12a ribonucleoproteins are also polar cohesin barriers, indicating that stalling may be intrinsic to cohesin itself. Finally, we show that RNA-DNA hybrids (R-loops) block cohesin-mediated DNA compaction in vitro and are enriched with cohesin subunits in vivo, likely forming TAD boundaries.

粘连蛋白和 CCCTC 结合因子 (CTCF) 是三维 (3D) 基因组组织的关键调节蛋白。 Cohesin 挤出由 CTCF 以极性方向锚定的 DNA 环。在这里，我们提供了 CTCF 结合极性控制粘连蛋白介导的 DNA 环的直接证据。使用单分子成像，我们证明 CTCF 的关键 N 末端基序可阻断粘连蛋白易位和 DNA 成环。粘连蛋白-CTCF复合物的冷冻电镜结构表明，当CTCF的N末端面向粘连蛋白时，锌指前面的CTCF基序只能到达其在STAG1粘连蛋白亚基上的结合位点。值得注意的是，C 端定向的 CTCF 通过粘连蛋白加速 DNA 压缩。 DNA 结合的 Cas9 和 Cas12a 核糖核蛋白也是极性粘连蛋白屏障，表明停滞可能是粘连蛋白本身固有的。最后，我们发现 RNA-DNA 杂合体（R 环）在体外阻断粘连蛋白介导的 DNA 压缩，并在体内富含粘连蛋白亚基，可能形成 TAD 边界。