RESUMEN
Nucleosomes containing the histone H3 variant CENP-A are the epigenetic mark of centromeres, the kinetochore assembly sites required for chromosome segregation. HJURP is the CENP-A chaperone, which associates with Mis18α, Mis18ß, and M18BP1 to target centromeres and deposit new CENP-A. How these proteins interact to promote CENP-A deposition remains poorly understood. Here we show that two repeats in human HJURP proposed to be functionally distinct are in fact interchangeable and bind concomitantly to the 4:2:2 Mis18α:Mis18ß:M18BP1 complex without dissociating it. HJURP binds CENP-A:H4 dimers, and therefore assembly of CENP-A:H4 tetramers must be performed by two Mis18αß:M18BP1:HJURP complexes, or by the same complex in consecutive rounds. The Mis18α N-terminal tails blockade two identical HJURP-repeat binding sites near the Mis18αß C-terminal helices. These were identified by photo-cross-linking experiments and mutated to separate Mis18 from HJURP centromere recruitment. Our results identify molecular underpinnings of eukaryotic chromosome inheritance and shed light on how centromeres license CENP-A deposition.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteína A Centromérica/metabolismo , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Sitios de Unión , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteína A Centromérica/química , Proteína A Centromérica/genética , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Células HeLa , Histonas/química , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Unión Proteica , Interferencia de ARN , Homología de Secuencia de AminoácidoRESUMEN
Centromeres are unique chromosomal loci that promote the assembly of kinetochores, macromolecular complexes that bind spindle microtubules during mitosis. In most organisms, centromeres lack defined genetic features. Rather, they are specified epigenetically by a centromere-specific histone H3 variant, CENP-A. The Mis18 complex, comprising the Mis18α:Mis18ß subcomplex and M18BP1, is crucial for CENP-A homeostasis. It recruits the CENP-A-specific chaperone HJURP to centromeres and primes it for CENP-A loading. We report here that a specific arrangement of Yippee domains in a human Mis18α:Mis18ß 4:2 hexamer binds two copies of M18BP1 through M18BP1's 140 N-terminal residues. Phosphorylation by Cyclin-dependent kinase 1 (CDK1) at two conserved sites in this region destabilizes binding to Mis18α:Mis18ß, limiting complex formation to the G1 phase of the cell cycle. Using an improved viral 2A peptide co-expression strategy, we demonstrate that CDK1 controls Mis18 complex recruitment to centromeres by regulating oligomerization of M18BP1 through the Mis18α:Mis18ß scaffold.