Dynamics of CENP-N kinetochore binding during the cell cycle

Abstract

SummaryAccurate chromosome segregation requires the assembly of kinetochores, multiprotein complexes that assemble on the centromere of each sister chromatid. A key step in this process involves binding of the constitutive centromere-associated network (CCAN) to CENP-A, the histone H3 variant that constitutes centromeric nucleosomes. This network is proposed to operate as a persistent structural scaffold for assembly of the outer kinetochore during mitosis. Here, we show by fluorescence resonance energy transfer (FRET) that the N-terminus of CENP-N lies in close proximity to the N-terminus of CENP-A in vivo, consistent with in vitro data showing direct binding of CENP-N to CENP-A. Furthermore, we demonstrate in living cells that CENP-N is bound to kinetochores during S phase and G2, but is largely absent from kinetochores during mitosis and G1. By measuring the dynamics of kinetochore binding, we reveal that CENP-N undergoes rapid exchange in G1 until the middle of S phase when it becomes stably associated with kinetochores. The majority of CENP-N is loaded during S phase and dissociates again during G2. We propose a model in which CENP-N functions as a fidelity factor during centromeric replication and reveal that the CCAN network is considerably more dynamic than previously appreciated.

A recently evolved class of alternative 3′-terminal exons involved in cell cycle regulation by topoisomerase inhibitors

“…This is consistent with the fact that CENPN association with centromeres is stabilized by its C-terminal domain ( Supplementary Fig. 3d, 'GFP-N DC-ter') [30][31][32] .…”

“…3d and Supplementary Fig. 3d, 'GFP-N12'), in addition to some diffuse staining as previously reported 30,32 . In contrast, the CENPN-pA13 isoform did not colocalize with CENPA or CENPH ( Fig.…”

The distinct functions of CENP-C and CENP-T/W in centromere propagation and function inXenopusegg extracts

“…14,15 Importantly, while CENP-A nucleosomes are stably bound to centromeric chromatin, 16 the binding of CCAN proteins appears to be dynamic. [17][18][19] CENP-C and CENP-T serve as a platform for recruitment of the KMN network through pathways that are not yet well understood. [20][21][22][23][24][25][26][27][28][29] From the CCAN components, CENP-N and CENP-C recognize CENP-A.…”

The Centromere: Epigenetic Control of Chromosome Segregation during Mitosis

“…However, CENP-N Chl4 is not required to sustain previously established centromeres, suggesting it may function redundantly at existing centromeres. In addition, centromeric CENP-N protein levels decrease before mitosis, suggesting that CENP-N may not be required to recruit kinetochore proteins (McClelland et al 2007;Hellwig et al 2011). Although CENP-N binds the CATD, this may not be necessary for CENP-N to localize to centromeres; centromeres generated by LacI-CENP-C and LacI-CENP-T fusions, which are proposed to be negative for CENP-A, are still positive for CENP-N (Gascoigne et al 2011), and CENP-N can be recruited to H3/CENP-A chimeras that lack the CATD in vitro (Guse et al 2011).…”

“…CENP-S and CENP-X also assemble at centromeres during S/G 2 (Dornblut et al 2014). CENP-N is stabilized specifically at the end of S phase through an unknown mechanism (McClelland et al 2007;Hellwig et al 2011). CENP-C and CENP-H are stabilized at centromeres during DNA replication (Hemmerich et al 2008).…”