Raf1 Is the DCAF of the CLRC Complex Figure 2. Raf1 is the DCAF for CLRC with WDxR motifs essential for heterochromatin formation. (A) Structural alignment of Raf1 (blue) with human DCAF DDB2 (grey). (B) Structural model of Rik1 (based on DDB1: grey) associated with Raf1 (red, green and yellow). In Raf1, the N- terminal helix (red) and the b-propeller (green and yellow) make specific interaction with Rik1. (C) Centromere silencing assay. Position of ade6+ marker gene in cen1. Wild-type cells with silenced cen1:ade6+ form red colonies; loss of silencing causes white colonies. (D) Lagging chromosomes in anaphase. Representative images of fixed cells stained with DAPI (red) and anti-tubulin (green) and % anaphase cells with lagging chromosomes. (E) H3K9me2 ChIP; levels associated with cen-dg relative to act1+, normalised to wild-type. Error bars: standard deviation (SD). (F) Northern: unprocessed otr transcripts (arrows) in wt, clr4D, raf1-R518A and raf1-R576A cells. Loading control: rRNA. (*): rRNA background. (G) Northern: centromeric siRNAs in wt, clr4D, raf1-R518A and raf1-R576A cells. Loading control: snoRNA58. doi:10.1371/journal.pgen.1002499.g002 25uC (Figure 4C). This demonstrates that raf1-1 cells are unable to form heterochromatin without RNAi and indicates that hetero- chromatin is completely erased by the raf1-1 mutation at 36uC. Surprisingly, unlike clr4D and raf1D mutants, centromeric siRNAs are produced at wild-type levels in raf1-1 cells at both temperatures (Figure 4D, Figure S4E and S5D). It is possible that siRNA levels remain high because raf1-1 inhibits the degradation of pre-existing siRNAs. However, high siRNA levels remain in raf1-1 cells which have undergone 144 divisions at 36uC; pre- existing siRNA would be diluted out (Figure S4E). Thus, the continual synthesis of siRNAs from centromere repeat transcripts must be unaffected by the defect in raf1-1. Moreover, these siRNAs are loaded into Ago1/RITS as indicated by their association with FLAG-Ago1 (Figure 4E). We conclude that as with raf1-R518A, the raf1-1 mutation uncouples siRNA production from H3K9 methylation. Methylation of H3K9, but not siRNA production, depends on an intact CLRC complex The interaction between the adaptor DDB1 and its DCAFs is known to require intact WDxR motifs in the DCAF [32,33,34]. Our structural alignment predicts that the raf1-1 T495I mutation is located on the ‘top’ surface of the WD-40 ring that interacts with Rik1 (Figure S5A). It is therefore possible that our specific raf1 mutations impair the ability of Raf1 to associate with Rik1 and that this interaction is essential for establishing and maintaining H3K9 methylation on heterochromatic loci. Indeed two-hybrid assays reveal that the binding of Raf1 to Rik1 [23] is disrupted in both WDxR motif mutants and the raf1-1 mutation (Figure 5A). All three mutations also impair co-immunoprecipitation of Rik1 with Raf1 from S. pombe extracts (Figure 5B). In addition, the association of the Clr4 H3K9 methyltransferase with Raf1 and Rik1 is lost in raf1-R518A, raf1-R576A and raf1-1 mutants (Figure 5C and Figure S5B). Recently we identified Stc1 as protein that links the RNAi machinery to CLRC [10]. Consistent with this, these same three specific raf1 mutations abolish the association of the Rik1 CLRC component with Stc1 (Figure S5C). Furthermore, ChIP analyses indicate that FLAG-Raf1-R518A, FLAG-Raf1-R576A and FLAG-Raf1-1 do not associate with centromeric repeats (Figure 5D). We conclude that binding of Raf1 to Rik1 is required to allow Raf1 to associate with centromeric repeats. This interaction is required to assemble an active CLRC complex in order to establish and maintain H3K9 methylation and hence functional heterochromatin. The coupling of H3K9 methylation and siRNA production could be achieved by bringing the distinct activities required for both processes together in the same complex. Since the raf1D and raf1-R576A mutations results in loss of siRNAs, Raf1 is clearly required for siRNA production (Figure S5D and Figure 2G). However, the raf1-R518A and raf1-1 mutants lose H3K9 methylation without affecting siRNA synthesis (Figure 2E, 2G; Figure 3C; Figure 4D; Figures S4E and S5D). This suggests that specific interactions between Raf1 and individual proteins in CLRC and/or other unknown factors are sufficient to allow siRNA generation in the absence of H3K9 methylation. An intact CLRC complex may not be required. Indeed, co-immunoprecip- itation experiments show that a weak but detectable interaction between Raf1 and Raf2 remains in raf1-1 and raf1-R518A, but not raf1-R576A cells (Figure 5E). We conclude that although an intact CLRC complex is dispensable for siRNA synthesis, a minimal Raf1-Raf2 interaction might be sufficient to allow the processing of centromere repeat transcripts to siRNAs. Discussion The interaction between the E3 ligase adaptor Rik1 and the DCAF Raf1 correlates with methylation of lysine 9 on histone H3 The CLRC complex, harbouring the histone methyltransferase Clr4Suvar3-9, performs an essential role in the establishment and maintenance of heterochromatic structures. In addition to Clr4, the complex contains the components Cul4, Rik1, Raf1 and Raf2. Rik1 is a WD-40 repeat protein that shares homology with the E3 ligase adaptor DDB1 [36]. DDB1 contains 21 WD-40 repeats that form the seven blades of three b-propellers that mediate association with Cul4 and DCAFs. This supports the possibility that Cul4-Rik1 form the core of an E3 ligase [24]. However, the C-terminus of Rik1 also shows homology with the b-propeller domain of cleavage and polyadenylation factor CSPF-A a well- known RNA binding protein [18]. Based on this CPSF-A homology, it has been suggested that Rik1 might bind RNA through this domain [21]. Although we cannot exclude that Rik1 is a bifunctional protein, our manual alignment detects 21 WD-40 repeats within Rik1 allowing it to be structurally aligned along its entire length with DDB1 (Figure 2B and Figure S2A). In our model, the CSPF-A homology domain corresponds to the b- propeller BPC involved in the interaction with the DCAF Raf1 (Figure 2B and Figure S2A). Moreover, we also show that Raf1 can be structurally aligned to the DCAF DDB2, the partner of DDB1 (Figure 2A, 2B and Figure S2B). As expected for a bona fide DCAF, Raf1 associates directly with the putative substrate adaptor Rik1 and contains two signature WDxR motifs which we show are required for the association of Raf1 with Rik1 and for H3K9 methylation and heterochromatin formation (Figure 2B and Figure S3). This suggests that the CLRC complex can adopt the architecture of a Cul4 E3 ligase in which Rik1 is the adaptor protein bridging the interaction between Cul4 and the DCAF Raf1 (Figure 6A). In addition, we show that the association of the DCAF Raf1 with Rik1 within an intact CLRC complex is critical for H3K9 methylation but it is dispensable for the processing of centromeric transcripts into siRNAs (Figure 2, Figure 4, and Figure 5). We envisage two alternative scenarios that can explain how the Cul4-Rik1-Raf1 complex might mediate H3K9 methylation (Figure 6A). First, it is possible that the CLRC is an active E3 ligase and that mono- or poly-ubiquitination of specific factors must occur to allow methylation of H3K9. Thus the DCAF Raf1 would be essential for the recognition and ubiquitination of key specific substrate(s) (Figure 6A top panel). In other systems, Cul4- PLoS Genetics | www.plosgenetics.org 6 February 2012 | Volume 8 | Issue 2 | e1002499