Dual phosphoregulatory mechanisms of condensin I revealed by biochemical reconstitution

Abstract Exactly how cell cycle regulators control sequential large-scale transformations of chromatin structure during mitosis is not fully understood. Here, through biochemical reconstitution with a minimal set of recombinant proteins, we demonstrate that the assembly and disassembly of mitotic chromatids are orchestrated by dual modes of phosphoregulation of condensin I. First, cyclin B-Cdk1 phosphorylates the terminal intrinsically disordered regions (tIDRs) of the non-structural maintenance of chromosome subunits, releasing condensin I from self-suppression imposed by these regions. Second, phosphorylation of a conserved residue in the central region of the kleisin subunit CAP-H by Cdk1 is essential for the full activation of condensin I. Conversely, addition of the protein phosphatase PP2A-B55 is sufficient to trigger the dissociation of condensin I from reconstituted chromatids, thereby driving their disassembly. Complementary analyses using Xenopus egg extracts reveal that the tIDRs and the kleisin central region are phosphorylated and dephosphorylated with distinct kinetics during mitotic entry and exit. Together, these findings uncover an elaborate yet streamlined regulatory network that tightly couples chromatid assembly and disassembly to mitotic progression.