A positive feedback mechanism ensures proper assembly of the functional inner centromere during mitosis in human cells.

The inner centromere region of a mitotic chromosome critically regulates sister chromatid cohesion and kinetochore-microtubule attachments. However, the molecular mechanism underlying inner centromere assembly remains elusive. Here, using CRISPR/Cas9-based gene editing in HeLa cells, we disrupted the interaction of Shugoshin 1 (Sgo1) with histone H2A phosphorylated on Thr-120 (H2ApT120) to selectively release Sgo1 from mitotic centromeres. Interestingly, cells expressing the H2ApT120-binding defective mutant of Sgo1 have an elevated rate of chromosome missegregation accompanied by weakened centromeric cohesion and decreased centromere accumulation of the chromosomal passenger complex (CPC), an integral part of the inner centromere and a key player in the correction of erroneous kinetochore-microtubule attachments. When artificially tethered to centromeres, a Sgo1 mutant defective in binding protein phosphatase 2A (PP2A) is not able to support proper centromeric cohesion and CPC accumulation, indicating that the Sgo1-PP2A interaction is essential for the integrity of mitotic centromeres. We further provide evidence indicating that Sgo1 protects centromeric cohesin to create a binding site for the histone H3-associated protein kinase Haspin, which not only inhibits the cohesin release factor Wapl and thereby strengthens centromeric cohesion but also phosphorylates histone H3 at Thr-3 to position CPC at inner centromeres. Taken together, our findings reveal a positive feedback-based mechanism that ensures proper assembly of the functional inner centromere during mitosis. They further suggest a causal link between centromeric cohesion defects and chromosomal instability in cancer cells.

HP1 cooperates with CAF-1 to compact heterochromatic transgene repeats in mammalian cells.

The nuclear organization of tightly condensed heterochromatin plays important roles in regulating gene transcription and genome integrity. Heterochromatic domains are usually present at chromosomal regions containing a large array of repeated DNA sequences. We previously showed that integration of a 1,000-copy tandem array of an inducible reporter gene into the genome of mammalian cells induces the formation of a highly compact heterochromatic domain enriched in heterochromatin protein 1 (HP1). It remains to be determined how these DNA repeats are packaged into a heterochromatic form and are silenced. Here, we show that HP1-mediated transgene condensation and silencing require the interaction with PxVxL motif-containing proteins. The chromatin assembly factor 1 (CAF-1) complex concentrates at the transgenic locus through the interaction of its PxVxL motif-containing p150 subunit with HP1. Knockdown of p150 relieves HP1-mediated transgene compaction and repression. When targeted to the transgenic locus, p150 mutants defective in binding HP1 cause transgene decondensation and activation. Taken together, these results suggest that HP1 cooperates with CAF-1 to compact transgene repeats. This study provides important insight into how heterochromatin is maintained at chromosomal regions with abundant DNA repeats.

WAC Promotes Polo-like Kinase 1 Activation for Timely Mitotic Entry.

The key mitotic regulator Polo-like kinase 1 (Plk1) is activated during G2 phase by Aurora A kinase (AurkA)-mediated phosphorylation of its activation loop, which is important for timely mitotic entry. The mechanism for Plk1 activation remains incompletely understood. Here, we report that the activation of Plk1 requires WAC, a WW domain-containing adaptor protein with a coiled-coil region that predominantly localizes to the nucleus in interphase. Cyclin-dependent kinase 1 (Cdk1) phosphorylates WAC, priming its direct interaction with the polo-box domain of Plk1. Knockdown of WAC compromises Plk1 activity and delays mitotic entry. These defects are rescued by exogenous expression of wild-type WAC, but not the Plk1-binding-deficient mutant. WAC also binds AurkA and can enhance Plk1 phosphorylation by AurkA in vitro. Taken together, these results indicate an important role for WAC in promoting Plk1 activation and the timely entry into mitosis.