Explorer The RFTS domain of Raf 2 is required for Cul 4 interaction and heterochromatin integrity in fission yeast

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-A 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. Citation: White SA, Buscaino A, Sanchez-Pulido L, Ponting CP, Nowicki MW, et al. (2014) The RFTS Domain of Raf2 Is Required for Cul4 Interaction and Heterochromatin Integrity in Fission Yeast. PLoS ONE 9(8): e104161. doi:10.1371/journal.pone. 0104161 Editor: Janet F. Partridge, St Jude Children’s Research Hospital, United States of America Received May 1, 2014; Accepted July 11, 2014; Published August 4, 2014 Copyright: 2014 White et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: The Wellcome Trust supported the work of R.C. A. (065061 and 095021) along with funding from the European Commission Network of Excellence EpiGeneSys (HEALTH-F4-2010-257082). The Wellcome Trust Centre for Cell Biology is supported by core funding from the Wellcome Trust (092076). RCA is a Wellcome Trust Principal Research Fellow. LSP and CPP were funded by the MRC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: robin.allshire@ed.ac. uk