Epigenetic engineering shows H3K4me2 is required for HJURP targeting and CENP-A assembly on a synthetic human kinetochore

EMBO J. 2011 Jan 19;30(2):328-40. doi: 10.1038/emboj.2010.329. Epub 2010 Dec 14.

Abstract

Kinetochores assemble on distinct 'centrochromatin' containing the histone H3 variant CENP-A and interspersed nucleosomes dimethylated on H3K4 (H3K4me2). Little is known about how the chromatin environment at active centromeres governs centromeric structure and function. Here, we report that centrochromatin resembles K4-K36 domains found in the body of some actively transcribed housekeeping genes. By tethering the lysine-specific demethylase 1 (LSD1), we specifically depleted H3K4me2, a modification thought to have a role in transcriptional memory, from the kinetochore of a synthetic human artificial chromosome (HAC). H3K4me2 depletion caused kinetochores to suffer a rapid loss of transcription of the underlying α-satellite DNA and to no longer efficiently recruit HJURP, the CENP-A chaperone. Kinetochores depleted of H3K4me2 remained functional in the short term, but were defective in incorporation of CENP-A, and were gradually inactivated. Our data provide a functional link between the centromeric chromatin, α-satellite transcription, maintenance of CENP-A levels and kinetochore stability.

Publication types

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

MeSH terms

  • Autoantigens / metabolism*
  • Centromere / metabolism
  • Centromere / physiology*
  • Centromere Protein A
  • Chromatin / genetics
  • Chromatin / metabolism*
  • Chromatin Immunoprecipitation
  • Chromosomal Proteins, Non-Histone / metabolism*
  • Chromosomes, Artificial, Human / genetics*
  • DNA Primers / genetics
  • DNA-Binding Proteins / metabolism*
  • Epigenesis, Genetic / genetics*
  • Genetic Engineering / methods
  • Histones / metabolism*
  • Humans
  • Kinetochores / metabolism
  • Nucleosomes / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction

Substances

  • Autoantigens
  • CENPA protein, human
  • Centromere Protein A
  • Chromatin
  • Chromosomal Proteins, Non-Histone
  • DNA Primers
  • DNA-Binding Proteins
  • Histones
  • Holliday junction recognizing protein, human
  • Nucleosomes