A Dual Inhibitory Mechanism Sufficient to Maintain Cell-Cycle-Restricted CENP-A Assembly

Abstract

Summary Chromatin featuring the H3 variant CENP-A at the centromere is critical for its mitotic function and epigenetic maintenance. Assembly of centromeric chromatin is restricted to G1 phase through inhibitory action of Cdk1/2 kinases in other phases of the cell cycle. Here, we identify the two key targets sufficient to maintain cell cycle control of CENP-A assembly. We uncovered a single phosphorylation site in the licensing factor M18BP1 and a cyclin A binding site in the CENP-A chaperone, HJURP, mediating specific inhibitory phosphorylation. Simultaneous expression of mutant proteins lacking these residues, results in complete uncoupling from the cell cycle. Consequently, CENP-A assembly is fully recapitulated under high Cdk activities, indistinguishable from G1 assembly. We find that Cdk-mediated inhibition is exerted by sequestering active factors away from the centromere. Finally, we show that displacement of M18BP1 from the centromere is critical for the assembly mechanism of CENP-A.

Posttranslational mechanisms controlling centromere function and assembly

“…Mis18BP1 and HJURP are the major targets of CDK phosphorylation in the CENP-A deposition pathway [51 •• ,52 •• ,59,62,63 •• ] (Figure 3a,b). The expression of Mis18BP1 containing mutations within the CDK phosphorylation sites leads to CENP-A loading in G2; however, the level of CENP-A deposited is much lower than that observed in G1 [51 •• ,59,63 •• ].…”

“…The expression of Mis18BP1 containing mutations within the CDK phosphorylation sites leads to CENP-A loading in G2; however, the level of CENP-A deposited is much lower than that observed in G1 [51 •• ,59,63 •• ]. Phosphorylation of sites within the Mis18α/β binding domain in the amino terminus of Mis18BP1, specifically Thr40 and Ser110, inhibit the binding of the Mis18α/β heterohexamer [51 •• ,52 •• ] (Figure 3b), providing a molecular basis of the inhibition of CENP-A deposition by CDK1 phosphorylation of Mis18BP1.…”

“…Phosphorylation of sites within the Mis18α/β binding domain in the amino terminus of Mis18BP1, specifically Thr40 and Ser110, inhibit the binding of the Mis18α/β heterohexamer [51 •• ,52 •• ] (Figure 3b), providing a molecular basis of the inhibition of CENP-A deposition by CDK1 phosphorylation of Mis18BP1. Phosphorylation of Thr653 also inhibits recruitment of the Mis18 complex to centromeres, but lies outside of the Mis18α/β interaction domain [63 •• ], suggesting there is more to be learned about how phosphorylation by CDK1 inhibits this complex.…”

Posttranslational modifications of CENP-A: marks of distinction

“…HJURP recognizes the CENP-A CATD (CENP-A targeting domain) through its N-terminal SCM3 homology domain and facilitates the loading of CENP-A-containing nucleosomes into DNA (Barnhart et al 2011; Bassett et al 2012; Dunleavy et al 2009; Foltz et al 2009; Jansen et al 2007; Sanchez-Pulido et al 2009; Shuaib et al 2010). CENP-A deposition is regulated by the phosphorylation of its deposition machinery by two kinases, PLK1 and CDK1/2 (McKinley and Cheeseman 2014; Silva et al 2012; Stankovic et al 2017). While PLK1 is a positive regulator of CENP-A deposition, CDK1/2 is inhibitory.…”

“…Mis18BP1 is a substrate for CDK1 phosphorylation, and modification of the protein disrupts the association of the protein with the Mis18 α / β hexamer (Pan et al 2017; Silva et al 2012; Spiller et al 2017). CDK1 also phosphorylates HJURP to inhibit CENP-A deposition (Muller et al 2014; Stankovic et al 2017). Thus the negative regulation by CDK1 ensures CENP-A deposition does not occur in G 2 , but is restricted to early G 1 , immediately following satisfaction of the mitotic checkpoint in human cells.…”