Assembly principles and stoichiometry of a complete human kinetochore module

Abstract

Centromeres are epigenetically determined chromosomal loci that seed kinetochore assembly to promote chromosome segregation during cell division. CENP-A, a centromere-specific histone H3 variant, establishes the foundations for centromere epigenetic memory and kinetochore assembly. It recruits the constitutive centromere-associated network (CCAN), which in turn assembles the microtubule-binding interface. How the specific organization of centromeric chromatin relates to kinetochore assembly and to centromere identity through cell division remains conjectural. Here, we break new ground by reconstituting a functional full-length version of CENP-C, the largest human CCAN subunit and a blueprint of kinetochore assembly. We show that full-length CENP-C, a dimer, binds stably to two nucleosomes and permits further assembly of all other kinetochore subunits in vitro with relative ratios closely matching those of endogenous human kinetochores. Our results imply that human kinetochores emerge from clustering multiple copies of a fundamental module and may have important implications for transgenerational inheritance of centromeric chromatin.

The Aurora B gradient sustains kinetochore stability in anaphase

“…Two mechanisms each maintain up to 50% of Mis12C (containing Dsn1) at kinetochores prior to anaphase: binding to CENP-C or CENP-T ( Huis In ’t Veld et al., 2016 ; Suzuki et al., 2015 ). The phosphorylation of Dsn1 at S100 and/or S109 by Aurora B is required for the stable association of Mis12C with both CENP-C and CENP-T ( Akiyoshi et al., 2013 ; Kim and Yu, 2015 ; Rago et al., 2015 ; Walstein et al., 2021 ; Yang et al., 2008 ). Dsn1 S100/S109 phosphorylation relieves the inhibitory effect of a basic region of Dsn1 on the interaction with CENP-C and CENP-T ( Dimitrova et al., 2016 ; Hara et al., 2018 ; Kim and Yu, 2015 ; Petrovic et al., 2016 ; Walstein et al., 2021 ).…”

“…The phosphorylation of Dsn1 at S100 and/or S109 by Aurora B is required for the stable association of Mis12C with both CENP-C and CENP-T ( Akiyoshi et al., 2013 ; Kim and Yu, 2015 ; Rago et al., 2015 ; Walstein et al., 2021 ; Yang et al., 2008 ). Dsn1 S100/S109 phosphorylation relieves the inhibitory effect of a basic region of Dsn1 on the interaction with CENP-C and CENP-T ( Dimitrova et al., 2016 ; Hara et al., 2018 ; Kim and Yu, 2015 ; Petrovic et al., 2016 ; Walstein et al., 2021 ). The interaction of Mis12C with CENP-T, but not with CENP-C, is additionally dependent on phosphorylation of CENP-T by Cyclin B-Cdk1 ( Gascoigne and Cheeseman, 2013 ; Gascoigne et al., 2011 ; Hara et al., 2018 ; Huis In ’t Veld et al., 2016 ; Nishino et al., 2013 ; Walstein et al., 2021 ).…”

“…Dsn1 S100/S109 phosphorylation relieves the inhibitory effect of a basic region of Dsn1 on the interaction with CENP-C and CENP-T ( Dimitrova et al., 2016 ; Hara et al., 2018 ; Kim and Yu, 2015 ; Petrovic et al., 2016 ; Walstein et al., 2021 ). The interaction of Mis12C with CENP-T, but not with CENP-C, is additionally dependent on phosphorylation of CENP-T by Cyclin B-Cdk1 ( Gascoigne and Cheeseman, 2013 ; Gascoigne et al., 2011 ; Hara et al., 2018 ; Huis In ’t Veld et al., 2016 ; Nishino et al., 2013 ; Walstein et al., 2021 ). To determine whether, like Dsn1 S109ph, total Dsn1 is also found in a gradient, we compared the distribution of phosphorylated and total Dsn1 in HeLa cells undergoing normal anaphase.…”

“…However, it is striking that key processes that control kinetochore assembly and function are driven by the activity of Cyclin B-Cdk1, which strongly declines at the metaphase-to-anaphase transition ( Clute and Pines, 1999 ; Murray et al., 1989 ), and Aurora B, which dissociates from centromeres and transfers to the central spindle in anaphase ( Carmena et al., 2012 ). For example, the phosphorylation of the KMN (Knl1 complex, Mis12 complex, Ndc80 complex) protein Dsn1 at S100 and/or S109 by Aurora B is required for the Mis12 complex (Mis12C) to stably associate with the constitutive centromere-associated network (CCAN) proteins CENP-C and CENP-T ( Akiyoshi et al., 2013 ; Dimitrova et al., 2016 ; Hara et al., 2018 ; Kim and Yu, 2015 ; Petrovic et al., 2016 ; Rago et al., 2015 ; Walstein et al., 2021 ; Yang et al., 2008 ). However, it is widely believed that the function of Aurora B at kinetochores ceases in anaphase ( Asbury, 2017 ; Mirchenko and Uhlmann, 2010 ; Parry et al., 2003 ; Vázquez-Novelle and Petronczki, 2010 ).…”

Multi-site phosphorylation of yeast Mif2/CENP-C promotes inner kinetochore assembly

“…The modified Mif2-PEST residues are not obviously conserved at the primary sequence level. Nevertheless, many or all Mif2/CENP-C homologues contain an analogous PEST region (Walstein et al, 2021). Clustered phosphorylation sites are common features of proteins with cell cycle-dependent functions (Nash et al, 2001;O'Neill et al, 1996;Serber and Ferrell, 2007).…”