Raf1 Is the DCAF of the CLRC Complex Figure 1. Raf1 is a chromatin-associated CLRC component. (A) Immunolocalisation of GFP-Raf1 (top panel) or untagged control (bottom panel) localisation. Representative images of fixed cells: GFP-Raf1 (green), centromere specific protein CENP-ACnp1 (red), DAPI stained DNA (blue). (B) FLAG-Raf1 ChIP. Diagram shows position of cen1 primers used (black bar). FLAG-Raf1 or untagged controls cells were analysed by multiplex PCR (otr enrichment relative to fbp1+ control - left) or by qPCR (otr enrichment relative to act1+ - right). I: input; IP: immunoprecipitation. (C) Proteins reproducibly detected in FLAG-Raf1 IPs by LC-MS/MS. Average peptide numbers identified in replicas is shown. (D) Raf1-FLAG IP analysed with anti- FLAG or with anti-Myc to detect Rik1-Myc. doi:10.1371/journal.pgen.1002499.g001 might represent the adaptor and the DCAF, respectively, for CLRC. To test these possibilities, we performed structural alignments of Rik1 and Raf1 and found that they can be modelled on DDB1 and on the DCAF DDB2 (Rik1: DOPE score 211385; Raf1: GA431 score 0.6 respectively) (Figure 2A and 2B). This model reveals that, like human DDB1, Rik1 is composed of three WD-40 b-propeller domains (BPA, BPB and BPC) composed of 216 WD-40 repeats and a C-terminal helical domain (Figure 2B and S2A). Raf1, like DDB2, is predicted to contain an N-terminal helical domain and a 7-bladed WD-40 b-propeller domain forming a ring (Figure 2A and 2B). Unlike DDB2, the helical and WD-40 domains of Raf1 are separated by an additional domain of unknown function (Figure S2B). As in the DDB1/ DDB2 complex, our model predicts that the Raf1 helical domain can be inserted into the Rik1 BPA-BPC cleft and the ‘top’ of the b- propeller ring interacts with the ‘bottom’ surface of Rik1 (Figure 2B). Alignment of the Raf1 WD-40 repeats with those of other known DCAFs revealed that Raf1 contains two WDxR motifs (1: aa515–518 and 2: aa573–576) (Figure S3A). WDxR motifs represent a ‘DCAF signature’ and are important for docking DCAFs to DDB1 [31,32,33,34]. Our Raf1 model indicates that these two WDxR motifs are located on the surface of the b- propeller ring and might provide specific sites for interactions with other factors (Figure S3E). To test the importance of Raf1 WDxR motifs, we mutated the endogenous raf1 gene in S. pombe to express FLAG-Raf1-R518A or FLAG-Raf1-R576A. Western analysis indicated that both mutant proteins are expressed at levels similar to wild-type FLAG-Raf1 (Figure S3B). All components of CLRC are required for heterochromatin integrity and consequently for the transcriptional silencing of marker genes placed within centromeric heterochromatin, and centromere function [6]. Like clr4D cells, heterochromatin-mediated silencing of cen1:ade6+ is disrupted in raf1-R518A and raf1-R576A mutants, as indicated by white/expressing rather than red/silent colonies (Figure 2C). Moreover, both mutations impair heterochromatin dependent silencing at the mating type locus (Figure S3D). Centromeric heterochromatin mediates robust sister-centro- mere cohesion and is therefore required for accurate chromosome segregation during mitosis [40,41]. Defective heterochromatin causes a quantifiable increase in the frequency of lagging chromosomes on late anaphase spindles [42]. Cells bearing the raf1-R518A or raf1-R576A mutation exhibit a frequency of lagging chromosomes equivalent to clr4D cells, indicating that this centromeric function of heterochromatin is disrupted (Figure 2D). Heterochromatin formation requires processing of centromeric transcripts into siRNA and methylation of H3K9 (H3K9me). Deletion of any gene encoding a CLRC component results in loss of H3K9me, accumulation of centromeric transcripts and a dramatic reduction of siRNA levels [17,18,19]. ChIP analyses indicate that H3K9me2 is reduced to background levels in raf1- R518A and raf1-R576A cells (Figure 2E). Importantly, Clr4 levels are equivalent to wild-type in both mutants (Figure S3C). Consistent with this loss of H3K9me2 and as observed in clr4D cells, high levels of centromeric transcripts also accumulate (Figure 2F). This is due to increased transcription given that higher levels of RNAPII are detected on centromeric repeats in both mutants compared to wild-type cells (Figure S5E). However, raf1-R576A cells contain low levels of centromeric siRNAs whereas raf1-R518A cells retain high levels of these siRNAs (Figure 2G and S3F). Our structural model predicts that R518 resides on the surface of Raf1 that interacts with Rik1 and predicts that the R518A mutation may specifically impair Raf1-Rik1 interactions (Figure S3G). In contrast, R576 is more distantly located from Rik1 and may have more profound structural effects. Together our findings demonstrate that the CLRC complex is likely to adopt a canonical Cul4-E3 ligase architecture and that Raf1 is a DCAF for CLRC. Moreover, we show that WDxR motifs are required for heterochromatin integrity and that the raf1- R518A mutation separates the function of CLRC in methylating H3K9 from its role in generating siRNAs. The raf1-1 mutation conditionally disrupts heterochromatin without affecting siRNA generation To dissect mechanisms governing heterochromatin assembly and maintenance, we screened for temperature-sensitive mutants that disrupt heterochromatin at the restrictive temperature (36uC) but not the permissive temperature (25uC) and isolated the raf1-1 mutation. raf1-1 produces a protein with a missense mutation (T495I) in the third WD-40 repeat; this residue is conserved in fungi suggesting that it is important for Raf1 function (Figure S3A). Similar levels of mutant FLAG-Raf1-1 and wild-type FLAG- Raf1 proteins are detected at 36uC thus phenotypes are not due to Raf1-1 degradation (Figure S4A). Marker gene silencing within centromeric repeats (cen1:ade6+ or cen1:ura4+), the mating type locus (mat3-M:ura4+) and at telomeres (tel1L:his3+) is alleviated in raf1-1 cells at 36uC, but not 25uC (Figure 3A and Figure S4B, S4C). Consistent with defective centromeric heterochromatin integrity, a high frequency of lagging chromosomes is observed in late anaphase raf1-1 cells at 36uC (Figure 3B). Moreover, H3K9me2 levels on centromeric repeats in raf1-1 cells are similar to wild-type cells at 25uC but negligible at 36uC (Figure 3C). Consistent with reduced H3K9me2, Swi6 localisation and unprocessed centro- mere repeat transcript accumulation is temperature dependent in raf1-1 cells (Figure 4A and 4B). Importantly, Clr4 levels are unaffected by the raf1-1 mutation (Figure S4B). Although we detect no H3K9me2 on centromere repeats in raf1-1 at 36uC, it is possible that residual levels of H3K9me2 remain. To determine if heterochromatin is erased at 36uC, a heterochromatin establishment assay was performed. At the mating type locus, active RNAi is required to establish heterochromatin, but once assembled, RNAi is not required to maintain heterochromatin [43]. Hence, loss of dcr1/RNAi has no impact on mating type locus silencing (i.e. mat3-M:ura4+). However, when presented with a naive template, dcr1D cells fail to assemble heterochromatin de novo and are unable to silence mat3-M:ura4+. To test whether the raf1-1 mutant erases hetero- chromatin structures, a raf1-1 dcr1D double mutant was generated at 36uC and mat3-M:ura4+ silencing assessed after shifting cells to 25uC. Silencing of mat3-M:ura4+ could not be established in this double mutant following a shift down from 36u to 25uC, but remained intact in the raf1-1 dcr1D double mutant generated at PLoS Genetics | www.plosgenetics.org 4 February 2012 | Volume 8 | Issue 2 | e1002499