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

Open Biol. 2014 Feb 12;4(2):130229. doi: 10.1098/rsob.130229.

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.

Keywords: centromere; constitutive centromere-associated network; kinetochore; mitosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis Regulatory Proteins / genetics
  • Apoptosis Regulatory Proteins / metabolism*
  • Binding Sites
  • Cell Line, Tumor
  • Centromere / metabolism*
  • Chromosomal Proteins, Non-Histone / genetics
  • Chromosomal Proteins, Non-Histone / metabolism
  • DNA / metabolism
  • DNA Damage
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Fluorescence Resonance Energy Transfer
  • G2 Phase
  • HeLa Cells
  • Histones / genetics
  • Histones / metabolism
  • Humans
  • Models, Molecular
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism*
  • S Phase
  • Tumor Suppressor Proteins / genetics
  • Tumor Suppressor Proteins / metabolism*

Substances

  • Apoptosis Regulatory Proteins
  • CENPS protein, human
  • CENPX protein, human
  • Chromosomal Proteins, Non-Histone
  • DNA-Binding Proteins
  • Histones
  • Nuclear Proteins
  • Tumor Suppressor Proteins
  • DNA