Distinct and compensatory roles of STAG1 and STAG2 in post-mitotic genome refolding

The 3D architecture of the eukaryotic genome is largely shaped by cohesin complexes containing either STAG1 or STAG2 subunits. Yet, their roles in post-mitotic genome refolding remain unclear. Here, we establish STAG2 as the predominant paralog and primary orchestrator of this process. We find that upon mitotic exit, STAG1 depletion imposes negligible effects on genome refolding or transcription reactivation, whereas STAG2 regulates genome remodeling in a stage- and chromatin-context-dependent manner. In early-G1, STAG2 promotes small euchromatic structural loops, enhancer-promoter contacts and transcriptional refiring; in late-G1, it suppresses large loops by limiting the more processive STAG1-cohesin. STAG2 processivity is constrained by CTCF roadblocks rather than genomic traveling distance. Co-depletion causes synergistic loss of structural loops and stronger transcriptional dysregulation, yet residual chromatin-bound cohesin retains measurable extrusion capacity. Together, these results establish STAG2 as the principal regulator of post-mitotic spatiotemporal chromatin reorganization, while STAG1 provides compensatory support for robustness.