Putting CENP-A in its place

Abstract

The centromere is the chromosomal region that directs kinetochore assembly during mitosis in order to facilitate the faithful segregation of sister chromatids. The location of the human centromere is epigenetically specified. The presence of nucleosomes that contain the histone H3 variant, CENP-A, are thought to be the epigenetic mark that indicates active centromeres. Maintenance of centromeric identity requires the deposition of new CENP-A nucleosomes with each cell cycle. During S-phase, existing CENP-A nucleosomes are divided among the daughter chromosomes, while new CENP-A nucleosomes are deposited during early G1. The specific assembly of CENP-A nucleosomes at centromeres requires the Mis18 complex, which recruits the CENP-A assembly factor, HJURP. We will review the unique features of centromeric chromatin as well as the mechanism of CENP-A nucleosome deposition. We will also highlight a few recent discoveries that begin to elucidate the factors that temporally and spatially control CENP-A deposition.

Fig. 1

a Primary sequences of human CENP-A and H3.1 are compared at single amino acid resolution. Dashes have been added at the relative position for H3.1. Known posttranslational modifications are mapped onto the sequence of both histones. Serine 7 phosphorylation by Aurora B in human cells is the only known post-translational modification on CENP-A. However, the serine at position seven is not a well-conserved feature of CENP-A even within vertebrates. Binding sites of HJURP, CENP-N, and CENP-C are highlighted on the CENP-A sequence, as well as the CENP-A-CENP-A dimerization domain (labeled nucleosome self-association). b Space-filling models of the CENP-A nucleosome as well as a CENP-A–CENP-A dimer from different perspectives (PDB ID: 3AN2). Highlighted amino acids are shaded to match the colors of the binding sites mapped in a. Two additional, non-conserved residues (R80, G81) in CENP-A constitute a bulge relative to H3

Fig. 2

Centromere and pericentromere chromatin organization depicted in interphase (1D model: left) and in mitosis (2D model: right). Histone H3 and CENP-A post-translational modifications are notated per cell cycle position, with black for constitutive modifications and red for mitosis-specific posttranslational modifications. In the 2D model, background shading distinguishes the organization of the centromere: pericentromere (dark grey), inner centromere (light grey), and outer centromere (pink, green/red)

Fig. 3

Overview of the cell cycle control mechanism of CENP-A deposition. The deposition of CENP-A is tightly regulated by the cell cycle. Chromosome schematics show the stepwise change in CENP-A protein levels at the centromere. a Starting at the exit from mitosis, each daughter centromere possesses one-half of the full complement of CENP-A nucleosomes (light pink oval). Cyclins are rapidly degraded following mitotic exit, and Cdk activity dramatically drops (dark blue gradient). Mis18BP1^KNL2 can interact with centromeric chromatin in its unphosphorylated state and primes the centromere for CENP-A deposition. b Once Mis18BP1^KNL2 associates with centromeres, HJURP is recruited and deposits newly synthesized CENP-A nucleosomes (light pink to red gradient oval). c In mid-to-late G1, the RSF complex (Rsf1-Snf2h) and MgcRacGap interact transiently with centromeres to stabilize newly assembly CENP-A nucleosomes and generate mature centromeric chromatin (red oval). By the S-phase transition, Cdk activity levels have increased above a threshold and are now unfavorable for Mis18 complex association with centromeres. Mis18BP1^KNL2 is phosphorylated, releases from chromatin, and CENP-A deposition is inhibited. d During S-phase, CENP-A nucleosomes are parceled to each daughter centromere (two light pink ovals)

Fig. 4

The deposition of newly synthesized CENP-A occurs during G1. The schematic shows a current model of CENP-A deposition into centromeric chromatin during the G1 phase of the cell cycle. Cdk activity decreases after the exit from mitosis, which allows the association of the Mis18 complex with centromeric chromatin, likely through an interaction with CENP-C. It is theorized that the Mis18 complex recruits chromatin modifying activity to centromeric histones in order to prime chromatin for the assembly of newly synthesized CENP-A by HJURP. Finally, the association of Rsf1/Snf2 and MgcRacGap with the centromere enables the establishment of fully stable CENP-A nucleosomes through chromatin remodeling and GTP-GDP of Cdc42 cycling

Fig. 5

Replication of centromeric chromatin and the S-phase dynamics of the most CENP-A-chromatin proximal CCAN proteins as the replication fork (RF) passes. Centromeric chromatin is replicated in S-phase concurrently with general chromatin. S-phase dynamics of the most CENP-A-chromatin proximal CCAN proteins: CENP-T/W/S/X complex, CENP-C, and CENP-N are also shown. Red arrows symbolize dissociation from the centromere. The green arrow symbolizes replication fork passage. Black arrows symbolize loading of new CENPs. a Existing CENP-A nucleosomes are allotted to each daughter strand, but no new CENP-A nucleosomes are added during S-phase. New H3.1/H3.3 nucleosomes may serve as placeholders during replication coupled dilution of existing CENP-A nucleosomes at the centromere (yellow nucleosomes). CENP-C is stably associated with centromeres in S-phase and likely tracts with CENP-A nucleosomes across the replication fork. b As the replication fork passes, CENP-T/W/S/X complexes are turned over every cell cycle, and load during late S-phase. CENP-N localization is dynamic throughout the cell cycle, but loads to maximal levels during S-phase

Fig. 6

The removal of non-centromeric CENP-A through the proteasome pathway. a Centromeric CENP-A is protected from degradation by binding partners that compete with the binding of CENP-A specific E3 ubiquitin ligases, such as Psh1. HJURP binding to unassembled heterodimers or specific interactions between centromeric CENP-A nucleosomes and the CCAN, inhibit the degradation of CENP-A. b Non-centromeric CENP-A is removed from ectopic locations in a cell cycle independent manner. This may occur as a natural consequence of histone exchange during chromatin remodeling and metabolism across chromosome arms or via a targeted degradation event by a specific E3 ubiquitin ligase, such as Psh1. The CENP-A specific E3 ubiquitin ligase has yet to be found in humans; however, research indicates that a CENP-A degradation can occur through the ubiquitin-mediated proteasome pathway