study are described in Table S1. Primer sequences are listed in Table S2. Structural modelling The homology model of Raf1 was created with the program Modeller (9v8) using the structure of DDB2 (PDB code 3EI4) [44]. Iterative rounds of alignment adjustment of the two protein sequences and model building were attempted until the Modeller scores (diagnostic of model quality) were optimised. The homology model of Rik1 was created with the program Modeller (9v8) using 5 templates (PDB code: 2B5L; 317N; 3189; 318C; 3E0C). The generated model (85.2) had the following model scores: RMSD: 1.536; MolPDF: 453.98; DOPE: 211385. Chromatin immunoprecipitation Chromatin immunoprecipitation (ChIP) was performed as described [54] with the following modifications. Cells were fixed in 1% PFA/15 min for H3K9me2 ChIP or in 1% PFA/20 min for RNA Polymerase II ChIP. For ChIP analyses of raf1-R518A and raf1-R576A strains, cells were grown at 32uC. For ChIP analyses of raf1-1 strain, cells were kept at the indicated temperature (25uC, 32uC or 36uC) for at least 96 hours. One microliter of monoclonal H3K9me2 antibody (m5.1.1), two microliter of anti-FLAG M2 monoclonal antibody (Sigma, F1804) or five microliters of RNA Polymerase II 8WG16 antibody (COVANCE, MMS-126R) was used per ChIP. Duplex PCR was performed to analyse ChIP samples using oligonucle- otides specific to the regions of interest and to the control gene fbp1 (Table S2). Real-time PCR (qPCR) was performed using the LightCycler 480 SYBR Green I Master (Roche) on a LightCycler 480 Instrument (Roche). qPCR analysis primers are in Table S2. Relative enrichments were calculated as the ratio of product of interest to control product (act1+ or tRNA) in IP over input. Histograms represent data from three biological replicates analysed in parallel. Immunoaffinity purification Immunoaffinity purifications (IP) for LC-MS/MS analysis were performed as described [55], with the following modifications: 5 g of cells were resuspended in ice-cold-lysis buffer (50 mM Hepes pH7.5, 150 mM KCl, 0.1% NP40). Immunoprecipitation was performed using proteinG Dynabeads resin (Life Technologies) coupled to anti-FLAG M2 antibody (Sigma, F1804) for 15 min. The IP’d material was treated with 500 U Benzonase, washed, subjected to on-bead Tryptic digestion, and prepared for LC-MS/ MS analysis as described previously [56]. Co-IPs for Western analysis were performed on 2 g of cells as above but for 1 hr. IP’d material was washed four times with ice- cold lysis buffer (50 mM Hepes pH7.5, 150 mM KCl, 0.1% NP40), resuspended in SDS sample buffer and analysed by SDS- PAGE. For tandem affinity purification (TAP)-tagged strains, Dynabeads coupled to IgG were used (gift from K. Hardwick). For Western analysis the following antibodies were used: anti-FLAG M2 (Sigma, F1804), anti-HA 12CA5 (gift from K. Samejima) and anti-myc (A14) (Santa Cruz sc-789), all at 1:1000. Further details of IP protocol are provided in Text S1. RNA analysis Northern analysis of long non-coding centromeric transcripts and centromeric siRNAs were performed as described previously [56]. RNA probes are listed in Table S2. Note: Further details of all experimental procedures are provided in Text S1. Raf1 Is the DCAF of the CLRC Complex Supporting Information Figure S1 Lid2-TAP is not pulled down with FLAG-Rik1. (A) Centromere silencing assay. Top: Position of ade6+ marker gene in cen1. Bottom: Wild-type cells with silenced cen1:ade6+ form red colonies. Cells expressing FLAG-Rik1 form red colonies. Loss of silencing results in white colonies, as observed in raf1D cells. (B) Lid2-TAP IP followed by western with FLAG antibody to detect Rik1-FLAG. I: 5% of input; IP: immunoprecipitation. (TIF) Figure S2 Rik1 contains 21 WD-40 repeats and Raf1 can adopt a DDB2-like structure. (A) Detection and comparison of WD-40 repeats in human DDB1 and Rik1. Alignment of DDB1 and Rik1 along with predicted structure. The 21 WD-40 repeats are highlighted in blue which were identified manually in Rik1 by comparison of secondary structures using PSIPRED. (B) Com- parison of DDB2 structure (left) with Raf1 model (right). Both proteins contain an N-terminal H-T-H (red) and a 7 bladed b- propeller (green). Unlike DDB2, Raf1 contains an additional domain (blue) of unknown function. (TIF) Figure S3 Raf1 contains two WDxR motifs important for protein function. (A) Representative multiple sequence alignment of the C-terminal WD-40 repeats of Raf1 in homologous proteins from fungi. Mutated residues mentioned in the text (T495I, R518A, R576A) are highlighted. (B) Western analyses of untagged, FLAG-Raf1, FLAG-Raf1R518A and FLAG-Raf1R576A from whole cell extracts. Wild-type and mutant Raf1 proteins (arrow) are expressed at similar levels. Asterisk *: indicates cross-reacting band which serves as a loading control. (C) Western analyses of untagged and Clr4-Myc in wild-type, raf1-R518A and raf1-R576A whole cell extracts. Loading control: Bip1. (D) Assay for silencing at mat3-M:ura4+. Plates are non-selective (N/S), lacking uracil (- URA) or supplemented with 5-FOA. Loss of silencing results in growth on -URA and loss of resistance to 5-FOA. (E) Top view of Raf1 b-propeller ring. Residues R518 and R576 are highlighted. (F) Northern: centromeric siRNAs in wild-type, clr4D, raf1-R518A and raf1-R576A cells. Loading control: snoRNA58. (G) Rik1-Raf1 model showing the position of residues R518 and R576. (TIF) Figure S4 raf1-1 impairs heterochromatin formation. (A) Western analyses of untagged, FLAG-Raf1 and FLAG-Raf1-1 from whole cell extracts. (B) Western analyses of Clr4-Myc in wild- type and raf1-1 whole cell extracts. A strain not expressing Clr4- Myc (no Myc) is included as a control. Loading control: Bip1. Cells were grown at 36uC. (C) Assay for silencing at mat3-M:ura4+. Plates are non-selective (N/S), lacking uracil (-URA) or supplemented with 5-FOA. Loss of silencing results in growth on -URA and loss of resistance to 5-FOA. (D) Assay for silencing at tel1L:his3+. Plates are non-selective (N/S) and lacking histidine (-HIS). Loss of silencing results in growth on –HIS. (E) Lagging chromosomes in anaphase wild-type and raf1-1 cells at 25uC or 36uC. Represen- tative images of fixed cells with DAPI (red) and anti-tubulin (green). (F) Northern: centromeric siRNAs in wild-type, clr4D, raf1- 1 cells. Cells were shifted from 25uC to 36uC for the indicated period of time (3, 6 and 12 days resulted in 36, 72 and 144 divisions, respectively, at the restrictive temperature). Loading control: snoRNA58. (TIF) Figure S5 raf1-1, raf1-R518A and raf1-R576A disrupts CLRC and heterochromatin integrity. (A) Rik1-Raf1 model showing the position of the T495I mutatedresidue in raf1-1. (B) Westerns of Rik1-FLAG IP analysed for Clr4-Myc in wild-type, raf1-R518A, PLoS Genetics | www.plosgenetics.org 12 February 2012 | Volume 8 | Issue 2 | e1002499