Human sister chromatids at metaphase are primarily linked by centromeric cohesion, forming the iconic X shape. Premature loss of centromeric cohesion disrupts orderly mitotic progression. Sgo1–PP2A) localizes to centromeres in mitosis, binds to Cohesin in a reaction requiring Cdk-dependent phosphorylation of Sgo1, dephosphorylates Cohesin-bound Sororin, and protects Cohesin at inner centromeres. The kinetochore kinase Bub1 phosphorylates histone H2A at T120 (H2A-pT120) and recruits Sgo1 to kinetochores, 0.5 µm from inner centromeres. Recently, we have shown that Sgo1 is a direct reader of the H2A-pT120 mark. Bub1 also recruits RNA polymerase II (Pol II) to unattached kinetochores and promotes active transcription at mitotic kinetochores. Pol II-dependent transcription enables kinetochore-bound Sgo1 initially recruited by H2A-pT120 to reach Cohesin embedded in centromeric chromatin (Figure 1). Our study demonstrates the importance of spatially constrained tug-of-war between opposing enzymatic activities in cell biology, and implicates mitotic transcription in targeting regulatory factors to highly compacted mitotic chromatin.
The ring-shaped Cohesin complex regulates transcription, DNA repair, and chromosome segregation by dynamically entrapping chromosomes to promote chromosome compaction and sister-chromatid cohesion. The Cohesin ring needs to open and close to allow its loading to and release from chromosomes. Recently, we have determined the crystal structures of the protease domain of Separase alone or in complex with an Scc1-derived peptide inhibitor. The structures reveal the structural basis of phosphorylation-enhanced Cohesin cleavage by Separase. In addition, we have determined the crystal structures of Wapl, Pds5B, and SA2/Scc3 bound to an Scc1 fragment. Our biochemical analyses further suggest that Pds5–Wapl stabilizes a transient, open state of cohesin to promote its release from chromosomes. Sororin inhibits a functional interaction between PDS5 and WAPL, thus stabilizing Cohesin on chromosomes.
The current available data collectively support the following model for Cohesin loading and release (Figure 2). Scc2–Scc4, Cohesin, and DNA form a transient complex, in which Scc2–Scc4 strengthens the engagement and ATP binding of the Smc1–Smc3 ATPase domains. ATP hydrolysis disengages the ATPase domains and opens the Cohesin ring to entrap DNA. ATP rebinding closes the inner gate and encircles DNA in the top Smc1–Smc3 closure. The entrapped DNA promotes ATP hydrolysis and inner-gate opening, allowing DNA to escape from the top closure. Wapl–Pds5 binds to the nucleotide-free Cohesin and, upon ATP binding, disrupts the Smc3–Scc1N interface to release DNA. Smc3 acetylation prevents the entrapped DNA from stimulating the ATPase activity of Cohesin, blocking inner-gate opening and Wapl–Pds5-dependent outer-gate opening.
Our ongoing research aims to determine the high-resolution structures of Cohesin in different nucleotide states, alone or bound to its regulators and DNA. In addition, we are investigating the mechanism by which cohesion establishment is coupled to DNA replication. These structural and functional studies will unveil the mystery behind the magic acts of DNA entrapment and release by the Cohesin ring.