In vitro centromere and kinetochore assembly on defined chromatin templates

Nature. 2011 Aug 28;477(7364):354-8. doi: 10.1038/nature10379.

Abstract

During cell division, chromosomes are segregated to nascent daughter cells by attaching to the microtubules of the mitotic spindle through the kinetochore. Kinetochores are assembled on a specialized chromatin domain called the centromere, which is characterized by the replacement of nucleosomal histone H3 with the histone H3 variant centromere protein A (CENP-A). CENP-A is essential for centromere and kinetochore formation in all eukaryotes but it is unknown how CENP-A chromatin directs centromere and kinetochore assembly. Here we generate synthetic CENP-A chromatin that recapitulates essential steps of centromere and kinetochore assembly in vitro. We show that reconstituted CENP-A chromatin when added to cell-free extracts is sufficient for the assembly of centromere and kinetochore proteins, microtubule binding and stabilization, and mitotic checkpoint function. Using chromatin assembled from histone H3/CENP-A chimaeras, we demonstrate that the conserved carboxy terminus of CENP-A is necessary and sufficient for centromere and kinetochore protein recruitment and function but that the CENP-A targeting domain--required for new CENP-A histone assembly--is not. These data show that two of the primary requirements for accurate chromosome segregation, the assembly of the kinetochore and the propagation of CENP-A chromatin, are specified by different elements in the CENP-A histone. Our unique cell-free system enables complete control and manipulation of the chromatin substrate and thus presents a powerful tool to study centromere and kinetochore assembly.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Autoantigens / chemistry
  • Autoantigens / metabolism
  • Cell Extracts
  • Cell-Free System
  • Centromere / metabolism*
  • Centromere Protein A
  • Chromatin / chemistry*
  • Chromatin / genetics
  • Chromatin / metabolism*
  • Chromosomal Proteins, Non-Histone / analysis
  • Chromosomal Proteins, Non-Histone / chemistry
  • Chromosomal Proteins, Non-Histone / metabolism
  • Chromosome Segregation
  • Conserved Sequence
  • Histones / metabolism
  • Humans
  • Kinetochores / metabolism*
  • Microtubules / metabolism
  • Mitosis
  • Oocytes
  • Protein Structure, Tertiary
  • Templates, Genetic
  • Xenopus laevis

Substances

  • Autoantigens
  • CENPA protein, human
  • Cell Extracts
  • Centromere Protein A
  • Chromatin
  • Chromosomal Proteins, Non-Histone
  • Histones