Raf1 Is a DCAF for the Rik1 DDB1-Like Protein and Has Separable Roles in siRNA Generation and Chromatin Modification Alessia Buscaino1, Sharon A. White1, Douglas R. Houston1, Erwan Lejeune1, Femke Simmer1¤a, Flavia de Lima Alves1, Piyush T. Diyora1, Takeshi Urano2, Elizabeth H. Bayne1¤b, Juri Rappsilber1, Robin C. Allshire1* 1 Wellcome Trust Centre for Cell Biology, School of Biological Sciences, The University of Edinburgh, Edinburgh, United Kingdom, 2 Department of Biochemistry, Shimane University Faculty of Medicine, Izumo, Japan Abstract Non-coding transcription can trigger histone post-translational modifications forming specialized chromatin. In fission yeast, heterochromatin formation requires RNAi and the histone H3K9 methyltransferase complex CLRC, composed of Clr4, Raf1, Raf2, Cul4, and Rik1. CLRC mediates H3K9 methylation and siRNA production; it also displays E3-ubiquitin ligase activity in vitro. DCAFs act as substrate receptors for E3 ligases and may couple ubiquitination with histone methylation. Here, structural alignment and mutation of signature WDxR motifs in Raf1 indicate that it is a DCAF for CLRC. We demonstrate that Raf1 promotes H3K9 methylation and siRNA amplification via two distinct, separable functions. The association of the DCAF Raf1 with Cul4-Rik1 is critical for H3K9 methylation, but dispensable for processing of centromeric transcripts into siRNAs. Thus the association of a DCAF, Raf1, with its adaptor, Rik1, is required for histone methylation and to allow RNAi to signal to chromatin. Citation: Buscaino A, White SA, Houston DR, Lejeune E, Simmer F, et al. (2012) Raf1 Is a DCAF for the Rik1 DDB1-Like Protein and Has Separable Roles in siRNA Generation and Chromatin Modification. PLoS Genet 8(2): e1002499. doi:10.1371/journal.pgen.1002499 Editor: Sue Biggins, Fred Hutchinson Cancer Research Center, United States of America Received July 20, 2011; Accepted December 9, 2011; Published February 2, 2012 Copyright: 2012 Buscaino 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. Funding: This research was supported by EMBO long-term fellowships to AB and FS, the Wellcome Trust (Senior Research Fellowship 084229/Z/07/Z to JR), and Principal Research Fellowship 065061/Z/01/A to RCA). The Centre for Cell Biology is supported by core funding from the Wellcome Trust (077707/Z/05/A and 092076/Z/10/Z). 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. * E-mail: robin.allshire@ed.ac.uk ¤a Current address: Department of Molecular Biology, NCMLS, Radboud University Nijmegen, Nijmegen, The Netherlands ¤b Current address: Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee, United Kingdom Introduction Silencing mechanisms mediated by small RNAs occur in most eukaryotes. RNA interference (RNAi) can reduce gene expression post-transcriptionally by cleaving homologous transcripts or by inhibiting their translation [1]. Small RNAs can also act in the nucleus, inducing transcriptional gene silencing (TGS) [2,3]. Although siRNA-directed modification of homologous chromatin is widespread in eukaryotes, the details of how siRNAs mediate such events remain limited in most systems. [4,5]. In plants and fungi, the link between siRNA-directed DNA/chromatin modifi- cation and heterochromatin formation is well established [2,3]. The fission yeast, Schizosaccharomyces pombe, is a powerful system in which to study RNAi-directed heterochromatin formation in part because it contains single non-essential genes encoding each of the key RNAi components. In fission yeast, siRNAs are important for heterochromatin formation on the centromeric outer repeats (composed of dg and dh elements) and other chromosomal locations. Although marker genes inserted at these loci are silenced [6], centromeric repeats are bi-directionally transcribed by RNAPII, producing double- stranded RNA (dsRNA) [2,3,7,8,9]. Dicer (Dcr1) cleaves these non-coding transcripts into siRNAs that guide the Argonaute (Ago1)-containing RITS complex to homologous nascent tran- scripts by sequence complementarity. Chromatin-bound-RITS recruits the histone methyltranferase Clr4SuVar3-9 (a CLRC component; see below) to centromeric repeats via the linker protein Stc1 [10]. Clr4 methylates lysine 9 of histone H3 (H3K9me), providing binding sites for the chromodomain proteins Swi6, Chp1 (a RITS component) and Chp2 [11,12,13,14]. Two different non-mutually exclusive models have been proposed to explain how heterochromatin, once assembled, silences centro- mere repeat transcription. In the ‘transcriptional gene silencing’ (TGS) model, heterochromatin factors directly repress transcrip- tion [15]. In the ‘co-transcriptional gene silencing’ (CTGS) model the repeats are continuously transcribed and silencing is due to the efficient processing of transcripts to siRNA [16]. In wild-type cells, siRNA production and H3K9 methylation are coupled processes: deletion of genes encoding CLRC components results not only in loss of H3K9 methylation but also in loss of siRNA [17,18,19]. Similarly, deletion of genes encoding RNAi components abrogates siRNA production and reduces H3K9me [9]. However, cells expressing only mutant histone H3 (H3K9R) produce some detectable siRNAs, even though H3K9 can not be methylated [20,21]. Moreover, tethering the Rik1 CLRC component to ura4 RNA triggers silencing of the ura4+ gene independently of other PLoS Genetics | www.plosgenetics.org 1 February 2012 | Volume 8 | Issue 2 | e1002499