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

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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, that mediated 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.

Fundação para a Ciência e a Tecnologia grants: (SFRH/BD/51878/2012, SFRH/BD/74284/2010, BIA-BCM/100557/2008); NIH/National Institute of General Medical Sciences grants: (R01-GM082989; T32-GM008275); NIH/NCI grant: (F30-CA186430); NIH grants (GM 037537, GM 110174); EMBO installation grant: (1818); ERC-consolidator grant: (ERC-2013-CoG-615638).