The RFTS domain of Raf2 is required for Cul4 interaction and heterochromatin integrity in fission yeast

PLoS One. 2014 Aug 4;9(8):e104161. doi: 10.1371/journal.pone.0104161. eCollection 2014.

Abstract

Centromeric heterochromatin assembly in fission yeast is critical for faithful chromosome segregation at mitosis. Its assembly requires a concerted pathway of events whereby the RNA interference (RNAi) pathway guides H3K9 methylation to target sequences. H3K9 methylation, a hallmark of heterochromatin structure, is mediated by the single histone methyltransferase Clr4 (equivalent to metazoan Suv3-9), a component of the CLRC complex. Loss of or defects in CLRC components disrupts heterochromatin formation due to loss of H3K9 methylation, thus an intact, fully functional CLRC complex is required for heterochromatin integrity. Despite its importance, little is known about the contribution of the CLRC component Raf2 to H3K9 methylation and heterochromatin assembly. We demonstrate that Raf2 is concentrated at centromeres and contrary to other analyses, we find that loss of Raf2 does not affect CENP-ACnp1 localisation or recruitment to centromeres. Our sequence alignments show that Raf2 contains a Replication Foci Targeting Sequence (RFTS) domain homologous to the RFTS domain of the human DNA methyltransferase DNMT1. We show that the Raf2 RFTS domain is required for centromeric heterochromatin formation as its mutation disrupts H3K9 methylation but not the processing of centromeric transcripts into small interfering RNAs (siRNAs) by the RNAi pathway. Analysis of biochemical interactions demonstrates that the RFTS domain mediates an interaction between Raf2 and the CLRC component Cul4. We conclude that the RFTS domain of Raf2 is a protein interaction module that plays an important role in heterochromatin formation at centromeres.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Centromere / genetics
  • Chromosome Segregation / genetics
  • Cullin Proteins / genetics*
  • Cullin Proteins / metabolism
  • DNA (Cytosine-5-)-Methyltransferase 1
  • DNA (Cytosine-5-)-Methyltransferases / genetics
  • DNA Methylation / genetics
  • Heterochromatin / genetics*
  • Histone Methyltransferases
  • Histone-Lysine N-Methyltransferase / genetics
  • Humans
  • Mitosis / genetics
  • Point Mutation
  • Protein Structure, Tertiary
  • RNA, Small Interfering
  • Schizosaccharomyces / genetics*
  • Schizosaccharomyces pombe Proteins / genetics*
  • Schizosaccharomyces pombe Proteins / metabolism
  • Sequence Alignment

Substances

  • Cullin Proteins
  • Heterochromatin
  • RNA, Small Interfering
  • Raf2 protein, S pombe
  • Schizosaccharomyces pombe Proteins
  • Histone Methyltransferases
  • DNA (Cytosine-5-)-Methyltransferase 1
  • DNA (Cytosine-5-)-Methyltransferases
  • DNMT1 protein, human
  • Histone-Lysine N-Methyltransferase