Retention of CENP‐A nucleosomes during DNA replication

Abstract

Centromeres are chromosomal loci that define the site of kinetochore formation and ensure equal chromosome segregation during mitosis and genomic stability. Centromeric identity is epigenetically regulated by the incorporation of the nucleosome containing the centromere specific CENP‐A histone H3 variant. In contrast to the canonical H3.1, CENP‐A deposition is independent of DNA replication, and occurs in early G1. Therefore new CENP‐A deposition and retention of CENP‐A during S‐phase are key events in epigenetic inheritance. DNA replication presents a challenge for CENP‐A inheritance because nucleosomes are removed to allow passage of the replication fork. However, CENP‐A nucleosomes are stably retained across S‐phase. We hypothesize that CENP‐A retention at centromeres during S phase is facilitated by a unique mechanism that distinguishes CENP‐A containing nucleosomes from bulk histones. We fused CENP‐A to the promiscuous biotin ligase (BirA*) to chemically label and affinity purify proteins transiently associated with CENP‐A during S‐phase. SILAC labeling and Mass Spec were used to identify S‐phase specific interactions. This analysis identified known G1 coupled CENP‐A deposition proteins such as HJURP and Mis18BP1 as well as novel factors, as associating with CENP‐A during S‐phase. Therefore the CENP‐A deposition proteins appear to play dual roles in S‐phase and G1. We demonstrate that HJURP is required for the inheritance of existing CENP‐A nucleosomes during DNA replication. MCM2 subunit of the replicative helicase binds HJURP and directly interacts with CENP‐A. Our data suggests that mechanism of transmission of CENP‐A nucleosomes requires CENP‐A specific deposition machinery including HJURP together with the activity of MCM2.Support or Funding InformationAHA Predoctoral Fellowship