title: The RFTS domain of Raf2 is required for Cul4 interaction and heterochromatin integrity in fission yeast.
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.
authors.affiliation: Wellcome Trust Centre for Cell Biology and Institute of Cell Biology, School of Biological Sciences, The University of Edinburgh, Edinburgh, Scotland, United Kingdom.
authors.forename: Sharon A
authors.initials: SA
authors.lastname: White
authors.affiliation: School of Biosciences, Kent Fungal Group, University of Kent, Canterbury, Kent, United Kingdom.
authors.forename: Alessia
authors.initials: A
authors.lastname: Buscaino
authors.affiliation: MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom.
authors.forename: Luis
authors.initials: L
authors.lastname: Sanchez-Pulido
authors.affiliation: MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom.
authors.forename: Chris P
authors.initials: CP
authors.lastname: Ponting
authors.affiliation: Wellcome Trust Centre for Cell Biology and Institute of Cell Biology, School of Biological Sciences, The University of Edinburgh, Edinburgh, Scotland, United Kingdom.
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Nowicki
authors.affiliation: Wellcome Trust Centre for Cell Biology and Institute of Cell Biology, School of Biological Sciences, The University of Edinburgh, Edinburgh, Scotland, United Kingdom.
authors.forename: Robin C
authors.initials: RC
authors.lastname: Allshire
mesh_terms: D002503:Centromere / Q000235:genetics; D020090:Chromosome Segregation / Q000235:genetics; D044842:Cullin Proteins / Q000235:genetics* / Q000378:metabolism; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics; D019175:DNA Methylation / Q000235:genetics; D006570:Heterochromatin / Q000235:genetics*; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase / Q000235:genetics; D006801:Humans; D008938:Mitosis / Q000235:genetics; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering; D012568:Schizosaccharomyces / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D044842:Cullin Proteins; D006570:Heterochromatin; D034741:RNA, Small Interfering; C513898:Raf2 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins; D000076983:Histone Methyltransferases; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human; D011495:Histone-Lysine N-Methyltransferase
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