Author Summary Heterochromatin is a specialized form of chromatin which is frequently assembled on DNA sequences with little or no coding potential. Heterochromatin formation involves specific post-translational modifications of histone tails (e.g. methylation of histone H3 on lysine 9). In fission yeast, Schizosaccharomyces pombe, heterochromatin is found at centromeres, telomeres, and the mating type locus. Heterochromatin integrity at centromeres is important for normal chromosome segregation. The heterochromatin associated repeats at fission yeast centromeres are known to be transcribed, and these non-coding transcripts are processed into siRNAs. siRNA production is required for establishment and maintenance of H3K9 methylation. But H3K9 methylation itself is required for siRNA production. It is not known how these two processes are coupled. In this study we use structural modelling and genetic analyses to demonstrate that the heterochromatin component Raf1 plays an essential role in coupling H3K9 methylation and siRNA production. Our analyses show that the heterochro- matin factors Rik1 and Raf1 can be structurally aligned with Cul4-E3 ubiquitin ligase components DDB1 and DDB2, respectively. We show that specific mutations impair the association of Raf1 with Rik1 and prevent H3K9 methylation but not siRNA production. These functional studies provide mechanistic insights into how siRNA production and chromatin modification are inte- grated. CLRC subunits, but requires RNAi [21]. Thus CLRC itself, rather than its substrate H3K9, promotes siRNA production independently of H3K9. However, it is not known how CLRC integrates histone methylation with siRNA generation. CLRC is composed of the histone methyltransferase Clr4, the b- propeller protein Rik1, the cullin protein Cul4, the WD-40 protein Raf1 (Clr8/Cmc1/Dos1) and Raf2 (Clr7/Cmc2/Dos2), which contains a RFTS domain [17,22,23,24,25]. Although it is not known whether CLRC acts as an E3-ubiquitin ligase in vivo, CLRC purified from cells exhibits E3 ligase activity in vitro [24]. E3 ligases regulate a variety of biological processes by bridging the E2 ubiquitin conjugating enzyme to specific substrates, allowing their ubiquitination (for review see [26]). Histone methylation is frequently regulated by ubiquitination but the mechanistic details remain unknown [27]. The Cullin-RING ligases (CRLs) are the largest family of multi- subunit E3 ligases in eukaryotes and are formed by a neddylated member of the cullin family scaffold (CUL1-CUL5), a RING finger protein (RBX1 or RBX2) and an adaptor protein that bridges the CUL/RING scaffold to substrate receptors [28]. Amongst CRLs, CRL4 is known to ubiquitinate histones [29,30]. In the CRL4 complex, DDB1 acts as the adaptor. Affinity purification of DDB1 from human cells identified various WD-40 proteins as possible substrate receptors termed DCAFs (DDB1 and CUL4 Associated Factors) [31,32,33,34]. Several DCAFs (WDR5, RBP5 and EED) not only interact with DDB1 but are also members of histone methyltransferase complexes [31,32,33,34]. Although it is not known whether the interaction between these DCAFs and CUL4/DDB1 leads to ubiquitination of substrates, it might have important functional consequences since the knock- down of CUL4 or DDB1 reduces histone methylation [31]. Many DCAFs contain a specific WDxR motif that is important for their association with DDB1 [31,32,33,34]. In S. pombe, Cul4 associates with the canonical adaptor Ddb1 [35] and with Rik1 (which shares similarity with Ddb1; [36]) in Raf1 Is the DCAF of the CLRC Complex CLRC [17,22,23,24]. Both Cul4-Ddb1 complex and CLRC affect heterochromatin integrity and neddylation of Cul4 is required for H3K9 methylation of heterochromatic regions [22,37]. This suggests that E3 ligase activity is involved in heterochromatin formation. However, heterochromatin defects observed in neddy- lation-defective Cul4 (cul4-K680R) could arise either from impaired Cul4-Ddb1 activity or from defective CLRC function. Furthermore, although Rik1 exhibits some similarity to Ddb1 and thus might act as an adaptor, recent analyses suggest that Rik1 is primarily an RNA binding protein which associates with centromeric transcripts via its CPSF-A domain [21]. Thus, it remains to be determined how CLRC contributes to heterochro- matin integrity and whether CLRC acts as a DDB1-related E3 ligase which promotes heterochromatin formation in vivo. Here we demonstrate that the CLRC components Rik1 and Raf1 can be structurally aligned with the human adaptor DDB1 and its DCAF DDB2, respectively. Our analyses are consistent with Raf1 acting as a DCAF for CLRC that contributes to siRNA amplification and H3K9 methylation by two distinct and separable routes. Our findings provide mechanistic insights into how siRNA production is integrated with chromatin modification via CLRC. Results Raf1 is a chromatin associated component of the CLRC complex Components involved in heterochromatin formation are typically localised in distinct chromatin-associated foci [14,38]. In contrast, previous studies overexpressing GFP-Raf1 from a strong promoter showed that it is a nuclear protein with no obvious chromatin localisation [23], however, this might not reflect its true subcellular localisation. Indeed, the genome-wide distribution of Raf1 suggests that it interacts predominantly with heterochromatic loci [13]. To reassess Raf1 localisation, we examined cells expressing GFP-Raf1 from its native promoter and found that it decorates several distinct chromatin-associated foci, the largest of which lies adjacent to the clustered kinetochores (Figure 1A). ChIP confirmed that FLAG-Raf1 associates with centromeric repeats (Figure 1B). We conclude that Raf1 is a chromatin-associated protein concentrated at heterochromatin. Biochemical purification of CLRC components Rik1, Raf2 and Clr4 identified Raf1 as an interacting partner [17,22,24]. In contrast, two-step purification of TAP-Raf1 identified the histone demethylase Lid2 plus Cul4 and Rik1, but apparently not Raf2 and Clr4, known CLRC subunits [39]. This raises the possibility that Raf1 is a component of two distinct complexes: CLRC (Cul4/ Rik1/Raf1/Raf2/Clr4) and an alternative Lid2/Cul4/Rik1/Raf1 complex. To further investigate this, we affinity selected FLAG- Raf1 from cell lysates and identified associated proteins by mass spectrometry. Silencing assays indicate that FLAG-tagged Raf1 is functional (Figure S1A). Single step FLAG affinity purification is less stringent than the two-step TAP affinity purification and was performed in mild conditions to identify as many Raf1 interacting proteins as possible. Although we detect all CLRC components, Lid2 peptides were absent (Figure 1C). Moreover, while Rik1 coimmunoprecipitated with Raf1 (co-IP; Figure 1D), Rik1 could not be detected in Lid2-TAP IPs (Figure S1B). We conclude that Raf1 mainly associates with the known CLRC components. Raf1 is a DDB2-like WDxR protein In CRL4 complexes DDB1 is the adaptor that recruits WD-40- containing DCAF proteins [28]. In CLRC, Rik1 is a DDB1- related protein that interacts with Raf1 [23]. Thus, Rik1 and Raf1 PLoS Genetics | www.plosgenetics.org 2 February 2012 | Volume 8 | Issue 2 | e1002499