A CENP-S/X complex assembles at the centromere in S and G2 phases of the human cell cycle

Abstract

The functional identity of centromeres arises from a set of specific nucleoprotein particle subunits of the centromeric chromatin fibre. These include CENP-A and histone H3 nucleosomes and a novel nucleosome-like complex of CENPs -T, -W, -S and -X. Fluorescence cross-correlation spectroscopy and Förster resonance energy transfer (FRET) revealed that human CENP-S and -X exist principally in complex in soluble form and retain proximity when assembled at centromeres. Conditional labelling experiments show that they both assemble de novo during S phase and G2, increasing approximately three- to fourfold in abundance at centromeres. Fluorescence recovery after photobleaching (FRAP) measurements documented steady-state exchange between soluble and assembled pools, with CENP-X exchanging approximately 10 times faster than CENP-S (t1/2 ∼ 10 min versus 120 min). CENP-S binding to sites of DNA damage was quite distinct, with a FRAP half-time of approximately 160 s. Fluorescent two-hybrid analysis identified CENP-T as a uniquely strong CENP-S binding protein and this association was confirmed by FRET, revealing a centromere-bound complex containing CENP-S, CENP-X and CENP-T in proximity to histone H3 but not CENP-A. We propose that deposition of the CENP-T/W/S/X particle reveals a kinetochore-specific chromatin assembly pathway that functions to switch centromeric chromatin to a mitosis-competent state after DNA replication. Centromeres shuttle between CENP-A-rich, replication-competent and H3-CENP-T/W/S/X-rich mitosis-competent compositions in the cell cycle.

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

“…Thus, CENP-T could be assembled first onto replicated chromatin and later on recruit CENP-W/S/X. In vitro assays suggest the existence of multiple DNA binding sites in CENP-T and CENP-W. 28,38 While in human cells the assembly of the CENP-T/W/S/X complex on chromatin occurs in late S/G2 phase cells, 18,36 it may take place in mitosis in the egg extracts. Xenopus homologues of CENP-S and CENP-X are present in the database but no antibodies against them are available yet to test when they are recruited to centromeres.…”

“…37,38 How CENP-T/W/S/X are targeted to the centromere is currently unclear although it has been reported that their incorporation occurs in late S phase/G2 and requires CENP-A. 18,35,36,39 It is also unclear whether they contribute to CENP-A deposition.…”

“…33,34 CENP-T and CENP-W do not bind CENP-A but instead recognize H3 nucleosomes present in centromeric chromatin. 35,36 These two proteins together with CENP-S and CENP-X, all 4 containing histone-fold domains, have been proposed to form a nucleosome-like particle capable of supercoiling DNA in vitro. 37,38 How CENP-T/W/S/X are targeted to the centromere is currently unclear although it has been reported that their incorporation occurs in late S phase/G2 and requires CENP-A.…”

Stepwise unfolding supports a subunit model for vertebrate kinetochores

“…The CCAN remains associated with centromeric chromatin across the entire cell cycle (6)(7)(8). The CCAN includes CENP-A, CENP-C, and four multisubunit complexes: CENP-L/-N (9), CENP-H/-I/-K/-M (10), CENP-O/-P/Q-/R/-U (11), and CENP-T/-W/-S/-X (12)(13)(14)(15).…”

The Centromere: Epigenetic Control of Chromosome Segregation during Mitosis

“…CENP-T and CENP-W completely turn over during S phase, but increase in abundance in S/G 2 relative to G 1 (Prendergast 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).…”