the RNAi components Ago1 or Dcr1. Thus, Clr-C can target H3K9me2 to centromeric repeats independently of the RNAi pathway. This data, plus our finding that ago1-deficient cells retain significant levels of H3K9me2 on centromeric repeats shows that Ago1 and Ago1 bound priRNAs are not necessary for the initiation of assembly of centromeric heterochromatin. Instead, our data strongly indicates that RNAi-independent mechanisms function together with RNAi in the cooperative assembly of centromeric heterochromatin. Results Transient depletion of rik1+ causes defective heterochromatin establishment in tas3WG cells To precisely define the point of action of regulators of heterochromatin assembly, we have employed the tas3WG allele which disrupts the RITS complex. Transient gene depletion experiments in tas3WG cells previously showed that the H3K9 methyltransferase, Clr4, is required to establish centromeric heterochromatin when Ago1 is separated from Chp1-Tas3 [10]. Clr4 is a component of a large complex of proteins called Clr-C. Cells lacking Clr-C components lose centromeric heterochromatin [24–29], but the role of individual Clr-C components in heterochromatin assembly is poorly understood. We therefore assessed the point of action of Clr-C components in heterochro- matin establishment, using transient gene depletion experiments in the tas3WG background. Rik1 was the first protein identified in complex with Clr4 [30], and it resembles the UV DNA damage binding protein, UV- DDB1, including homology to the CPSF-A factor involved in RNA processing [31]. Rik1 is thought to act upstream of Clr4, and to help recruit Clr-C to chromatin, since Rik1 remains localized at centromeres in mutants that mislocalize Clr4 [5,28]. We tested whether transient depletion of rik1+ in tas3WG cells would prevent assembly of heterochromatin. We introduced the tas3WG-TAP and tas3-TAP alleles into a rik1D strain that carries a ura4+ transgene within the outer repeats of centromere 1 (cen::ura4+). Wild type cells efficiently assemble heterochromatin on cen::ura4+, silencing its expression, and allowing growth on media containing the drug 5- FOA, which is toxic to cells that express ura4+. Cells lacking rik1+ fail to silence the centromeric transgene. The re-establishment of centromeric heterochromatin was monitored following reintro- duction of rik1+ into its normal genomic locus. Addition of rik1+ to rik1D tas3-TAP cells allowed efficient establishment of heterochro- matin and silencing of the cen::ura4+ transgene (Figure 1B). In contrast, on reintroduction of rik1+ into tas3WG-TAP cells, heterochromatin did not reassemble to silence the cen::ura4+ reporter. Transcription of endogenous dg and dh centromeric repeats was measured by real time PCR in cDNA prepared from these strains. In wild type cells, centromeric transcripts are processed by siRNA- dependent Ago1-mediated processing and by RNAi-independent turnover [32–34]. In addition, heterochromatin that assembles on repeat sequences can reduce access of RNA polymerase, thus preventing transcript accumulation [35]. Centromeric transcripts from dh (Figure 1C) and dg (Figure 1D) accumulate in cells lacking rik1+, similar to cells lacking clr4+. On reintegration of rik1+ into tas3-TAP cells, centromeric transcripts become normally pro- cessed, resulting in no net gain in transcript levels in rik1D to rik1+ tas3-TAP cells relative to tas3-TAP cells. Strikingly, both dg and dh transcript levels remain high in tas3WG cells following reintegration of rik1+, consistent with the observed silencing defect of the cen::ura4+ reporter in these strains. This accumulation of transcripts is at least in part due to defective processing of centromeric Clr-C Required for Heterochromatin Establishment transcripts into siRNAs by the RNAi machinery since siRNAs were not detectable by Northern blotting in tas3WG-TAP rik1D to rik1+ cells whereas rik1+ reconstituted tas3-TAP cells synthesized centromeric siRNAs as efficiently as tas3-TAP cells (Figure 1E). Transient depletion of Raf1 or Raf2 in tas3WG cells causes defective heterochromatin assembly Raf1 (Cmc1, Dos1, Clr8) and Raf2 (Cmc2, Dos2, Clr7) have also been identified as components of Clr-C [27,29]. They are required for localization of Swi6 [25], and are important for silencing the mating type locus [26]. raf1+ encodes a WD repeat protein which can bind Rik1 [25], and raf2+ encodes a putative Zn finger protein which binds to Pcu4 [26]. raf1D and raf2D were crossed into tas3-TAP and tas3WG -TAP backgrounds, and wild type genomic copies of raf1+ or raf2+ were reintegrated into the corresponding deletion mutants and assessed for heterochromatin assembly. As seen for transient depletion experiments with rik1, tas3-TAP cells efficiently re-assembled centromeric heterochromatin on reintroduction of raf1+ or raf2+, whereas silencing of the cen::ura4+ reporter was not apparent in tas3WG-TAP backgrounds (Figure 2A and 2B). Centromeric transcripts accumulate to high levels in raf1 and raf2 deleted cells, and although transcript levels drop following reintegration of raf1+ into raf1D tas3-TAP cells or of raf2+ into raf2D tas3-TAP cells, high levels of dg and dh transcripts are maintained in both raf1+ and raf2+ reconstituted tas3WG-TAP cells (Figure 2C and 2E, Figure S1A and S1B). Consistent with this failure to suppress high levels of centromeric transcription in tas3WG-TAP cells transiently depleted for raf1+ or raf2+, these cells fail to engage the RNAi pathway to promote destruction of centromeric transcripts into siRNAs (Figure 2D and 2F). Transient depletion of Pcu4 in tas3WG cells causes defective heterochromatin assembly Pcu4 is the fission yeast cullin4, and it has been identified in complex with the UV-DDB1 E3 ubiquitin ligase [35,36], and with the related Rik1 protein in the Clr-C complex [27–29]. To define the role of Pcu4 in heterochromatin establishment, we monitored heterochromatin assembly following reintroduction of the pcu4+ gene into pcu4D tas3-TAP and tas3WG-TAP strains. Clearly centromeric transcripts accumulate in pcu4D cells, and processing of transcripts is efficiently resumed following re-introduction of the wild type gene into tas3-TAP cells (Figure 3A and 3B). However, both dh and dg transcript levels are maintained at high levels following pcu4+ reintroduction into pcu4D tas3WG -TAP cells. This failure to silence centromeric transcripts was reflected in the failure to produce abundant centromeric siRNAs in these tas3WG reconstituted cells (Figure 3C). In summary, all components of Clr-C are defective for silencing of endogenous centromeric or centromeric reporter transcripts following their transient depletion in tas3WG cells, suggesting that their continuous presence is required for the initiation of heterochromatin in RITS-defective cells. Defective heterochromatin assembly on transient depletion of Clr-C complex in tas3WG cells correlates with absence of centromeric H3K9me2 Next we analyzed H3K9 methylation on centromeric sequences following transient depletion of components of the Clr-C complex. In wild type cells, H3K9me accumulates to high levels on centromeric repeats through both RNAi-dependent and RNAi- independent mechanisms [15,30]. In cells lacking pcu4, H3K9me2 levels on dh sequences are not above the background seen for clr4D PLoS Genetics | www.plosgenetics.org 3 October 2010 | Volume 6 | Issue 10 | e1001174