Raf1 Is the DCAF of the CLRC Complex panel) or kept at 36uC (bottom panel). non-selective (N/S), –URA or 5-FOA plates indicated. Silencing (wt, dcr1D and raf1-1 at 25uC) allows little growth on -URA but good growth on 5-FOA. Loss of silencing (rik1D, raf1-1 at 36uC and raf1-1 dcr1D at 25u or 36uC) results in good growth on –URA and 5-FOA sensitivity. (D) Northern: centromeric siRNA in indicated cells at 25u or 36uC. Loading control: snoRNA58. (E) Northern: FLAG-Ago1- associated siRNA from indicated cells at 25u or 36uC. doi:10.1371/journal.pgen.1002499.g004 E3 ligases have been shown to ubiquitinate histones [29,30], it is possible that a specific histone residue needs to be ubiquitinated to allow methylation of H3K9. E3 ligase activity has been shown to associate with affinity selected CLRC in vitro [24], however, we have been unable to detect this activity in similar experiments. This suggests that CLRC E3 ligase activity may be particularly inefficient and/or the ubiquitination events that it mediates are transient. The alternative scenario is that the CLRC complex is not an active E3 ligase in vivo and that Cul4, Rik1, Raf1 and Raf2 just form a protein scaffold that acts to target Clr4 methyltransferase to heterochromatic repeats, independently of ubiquitination (Figure 6A bottom panel). In this case the specific mutations which impair the Rik1-Raf1 interaction may just disrupt the scaffold so that Raf1 is not targeted to the heterochromatic repeats and Clr4 no longer associates with Rik1 and Raf1. Interestingly, the DCAF DDB2 has been shown to bind DNA with the ‘bottom’ surface of its WD-40 ring [44]. Similarly, the WD-40 ring of the DCAF WDR5 specifically binds the histone H3 N-terminal tail methylated on lysine 4 [45,46,47]. Thus, it is possible that the WD-40 ring of the DCAF Raf1 also binds DNA or histones to allow the correct targeting of the histone methyltransferase Clr4 to heterochromatic repeats. Raf1 function in H3K9 methylation and RNAi are separable CLRC plays a dual role in heterochromatin formation: it harbours the histone methyltransferase Clr4 and hence it is responsible for H3K9 methylation, it also mediates siRNA production since cells lacking any single CLRC subunit have low centromeric siRNA levels (Figure S5D and [17,18]). Therefore, in wild-type fission yeast H3K9 methylation and siRNAs synthesis are coupled. This ensures formation of heterochromatin at centromeres, telomeres, and the mating type locus, and prevents promiscuous silencing at other chromosomal regions. However, cells expressing only mutant histone H3 (H3K9R) have been shown to produce some detectable siRNAs, even though H3K9 can not be methylated [20,21]. Such analyses implicate CLRC itself, rather than its known substrate H3K9, in promoting siRNA production independently of H3K9. Here we have isolated two mutations in the raf1 gene (raf1-1 and raf1R518A) that destroy CLRC integrity without affecting siRNA levels. Although we cannot exclude that a minimal CLRC complex is still present in these mutants, our results indicate that individual CLRC components can stimulate processing of centromeric transcripts into siRNAs independently of CLRC complex integrity (Figure 6B). It remains to be determined how CLRC components promote this H3K9 methylation-independent siRNA production. Our analyses of specific raf1 mutants suggests that the putative E3 ligase activity of the CLRC complex is not required for siRNA synthesis given that in raf1-1 and raf1-R518A mutants (predicted to impair the E3 ligase activity of the complex) siRNA levels remain high (Figure 2G and Figure 4D). One possibility is that Clr4 can methylate specific substrates indepen- dently of CLRC integrity and that this is a key event in siRNA production [21]. In accordance with this, Clr4 was recently shown to methylate the RNA processing factor Mlo3 and this methylation correlates with high siRNA levels [48]. However, Clr4-mediated methylation of specific substrates cannot be the only event required to trigger siRNA production since the loss of any CLRC component results in dramatic reduction of siRNA levels. We find that high siRNA levels correlate with a low but detectable Raf1- Raf2 interaction. Complete disruption of this Raf1-Raf2 interac- tion (as observed in raf1-R576A) cuts siRNA production to undetectable levels. Interestingly, centromeric repeats are tran- scribed preferentially in S-phase and Raf2 has been recently shown to interact with Cdc20 (the catalytic subunit of DNA- polymerase e) [49,50,51]. Coordination of DNA replication, siRNA generation and methylation of specific substrates may be essential for heterochromatin establishment and maintenance of heterochromatic structures. Our analyses suggest that integration of DNA replication, siRNA production and methylation of H3K9 is achieved by bringing the distinct activities required for these processes together in the same protein complex (CLRC). This ensures the assembly of robust heterochromatin structures. Cotranscriptional gene silencing plays a subsidiary role in repression of heterochromatic repeats Two different models have been proposed to explain how heterochromatin could silence centromeric repeats. In the ‘TGS’ model, heterochromatin factors have been proposed to repress transcription at centromeres [15] whereas the ‘CTGS’ model suggests that centromere repeats are continuously transcribed and silencing is caused by the efficient cleavage of centromeric transcripts by RNAi into siRNA [16]. Our analyses demonstrate that the RNAi machinery is active in raf1-1 cells, so that centromeric transcripts are processed into siRNAs independently of heterochromatin integrity. raf1-1 cells produce siRNAs at levels similar to wild-type cells and they are loaded into Ago1. However, despite this, high levels of unprocessed centromeric transcripts persist, indicating that RNAi fails to destroy them. The fact that heterochromatin is absent in raf1-1 and raf1-R518A cells and transcripts remain high, even though high levels of homologous Ago1-associated centromeric siRNAs are present, is more compatible with a TGS model where heterochromatin directly represses RNAPII transcription of centromere repeats. In agreement with this model, RNAPII ChIP clearly shows an increase of RNAPII occupancy in the raf1 mutants compared to wild-type cells (Figure S5E). This observation is also consistent with the fact that tethered Clr4 can generate heterochromatin and silence marker genes independently of RNAi [52]. The coupling of non-coding transcription with chromatin modification to establish and maintain distinct epigenetic states is a general mechanism of gene regulation in eukaryotes. Links between Cul4-dependent ubiquitination and histone methylation are continuing to emerge in different systems. Further analyses will determine how these activities are integrated to regulate specific chromatin states. Materials and Methods Strain and plasmid construction Standard procedures were used for bacterial and fission yeast growth and genetic manipulations [53]. S. pombe strains used in this PLoS Genetics | www.plosgenetics.org 9 February 2012 | Volume 8 | Issue 2 | e1002499