AT-hook-dependent DNA loop extrusion by STAG1 drives 3D genome folding

Cohesin, a ring-shaped complex composed of Smc1, Smc3, Scc1 and STAG, is essential for sister chromatid cohesion and the regulation of three-dimensional (3D) genome architecture. At the single-molecule level, cohesin extrudes DNA loops, a process thought to drive higher-order genome folding. In vertebrates, cohesin incorporates either STAG1 or STAG2. Although both support sister chromatid cohesion, they differentially regulate 3D genome organization. However, the mechanistic basis for these differences has remained unclear. Here we show, using single-molecule assays, that cohesin-STAG1 extrudes DNA loops more efficiently than cohesin-STAG2, despite comparable ATPase activity and topological DNA entrapment. We identify an AT-hook motif unique to the STAG1 N-terminus as the element that promotes loop extrusion without altering ATPase activity or DNA binding. In human somatic cells, the AT-hook is required for stable cohesin-chromatin association during G1 phase but is dispensable for sister chromatid cohesion. Mutation of this motif markedly impairs TAD and chromatin loop formation. These findings highlight AT-hook as a critical determinant that distinguishes STAG1 from STAG2 by promoting DNA loop extrusion and stabilizing cohesin-chromatin interactions in interphase through a mechanism distinct from the one underlying sister chromatid cohesion.