Author Summary Centromeres are the chromosomal regions that promote chromosome movement during cell division. They consist of repetitive DNA sequences that are packaged into heterochromatin. Disruption of centromeric heterochro- matin leads to chromosome loss that can result in miscarriages and genetic disorders. We have sought to define the precise steps leading to heterochromatin assembly using fission yeast as the model system. To accomplish this we employed our novel Tas3WG mutant strain that can propagate preassembled heterochromatin but cannot support its de novo establishment. Current models suggest that small RNAs initiate heterochromatin assembly by targeting the RNAi machinery and subse- quently the Clr-C chromatin-modifying complex to the centromere. Here, we demonstrate that transient deple- tion of components of the RNAi pathway that generate or bind small RNAs does not perturb heterochromatin assembly in our Tas3WG strain. Instead, transient depletion of the Clr-C complex blocks heterochromatin assembly, suggesting a critical role for continuous Clr-C activity during heterochromatin assembly in Tas3WG cells. We have directly tested whether Clr-C can target centromeres when expressed in cells deficient for RNAi and Clr-C. We find that RNAi–independent recruitment of Clr-C can occur and likely contributes to the critical initiating mechanisms of heterochromatin assembly. tion of RNA via cleavage mediated by Argonaute. In fission yeast, the RNAi effector complex is called RITS, and consists of the sole argonaute protein, Ago1, in complex with Tas3 which physically links Ago1 to Chp1 [8,10]. Association of the RITS complex with centromeres is co-dependent on the RNA dependent RNA polymerase complex, RDRC [11]. RITS and RDRC physically associate with the Clr4-containing Clr-C complex [5,12], and trigger an RNAi-mediated positive feedback loop to enhance H3K9me2 accumulation and heterochromatin assembly [13]. Accumulation of H3K9me2 allows recruitment of heterochroma- tin- binding proteins such as Swi6 (the fission yeast homolog of HP1) and cohesin to centromeric repeats, and is required for efficient chromosome segregation (reviewed in [14]). Clearly, the mechanism by which RITS and RDRC are initially recruited to centromeres to promote RNAi-dependent accumulation of H3K9me2 is critical to our understanding of heterochromatin assembly. Somewhat paradoxically, the outer repeats of the centromere are transcribed by RNA polymerase II, and this transcription correlates with heterochromatin assembly [15–18]. Recently, two models have been proposed for how centromeric transcripts may initiate recruitment of RITS/RDRC. The first proposes that single stranded centromeric transcripts fold into hairpin structures to provide dsRNA template for the activity of dicer (Dcr1) to generate centromeric siRNAs to target RITS to homologous centromeric sequences [19]. Alternatively, RNA degradation products (priRNAs) associate with Ago1, and if derived from centromeric transcripts, target Ago1 to centromeres [20]. Ago1 slices transcripts that are homologous to priRNAs, recruiting RDRC which promotes dsRNA synthesis. dsRNAs are cleaved by Dcr1 to form centromeric siRNAs that recruit RITS to centromeres [8]. In both models, centromeric RITS/RDRC then promotes Clr-C association, and H3K9 methylation facilitating binding of Chp1 to chromatin. These models infer that small RNAs and the RNAi pathway act as the priming signal for heterochromatin assembly, with Dcr1 or Clr-C Required for Heterochromatin Establishment Ago1 playing the initiating role respectively. However, RITS possesses two potential centromeric targeting motifs: Ago1 which binds siRNAs and can target centromeric transcripts and Chp1 which has high affinity for binding H3K9me2 [6,7]. We questioned whether indeed RNAi is the upstream event for heterochromatin assembly, or whether Clr4 functions upstream of RNAi to generate H3K9me2 to recruit RITS. Dissection of the requirements for the initial assembly of centromeric heterochromatin is greatly hampered by the inter- relatedness of the RNAi and chromatin modifying pathways and the positive feedback loops involved in full heterochromatin assembly. Deletion of genes required for assembly of heterochro- matin ablates heterochromatin, complicating analysis of whether the gene contributes to the establishment or maintenance of heterochromatin. To define the contribution of siRNAs and H3K9me to targeting RITS to centromeres, we have generated mutants that separate Ago1 from the RITS complex [10], that remove Chp1 from the RITS complex [21], and mutations within the chromodomain of Chp1 [6,22] that weaken the high affinity of Chp1’s chromodomain for binding H3K9me2/3. Data accumu- lated from analysis of these mutants strongly supports that centromeric targeting of RITS critically depends on Chp1 and in particular, Chp1 chromodomain’s high affinity for binding H3K9me2/3. The tas3WG mutant separates Ago1 from Tas3-Chp1 [10]. This mutant bears a two amino acid alanine substitution of residues W265 and G266 within the conserved WG/GW Ago ‘‘hook’’ (or interaction domain) of Tas3 [10,23], that renders the Chp1-Tas3 subcomplex incapable of associating with Ago1. Surprisingly, tas3WG cells can maintain preassembled heterochromatin, most likely via retention of the subcomplexes of RITS at centromeres because of Chp1-Tas3 association with H3K9me2 and Ago1’s association with centromeric siRNAs [10]. Interestingly, following removal of all H3K9me2 and loss of heterochromatin –dependent siRNAs (in clr4D backgrounds), tas3WG cells fail to generate de- novo heterochromatin on reintegration of clr4+ [10]. We reasoned that genes that are particularly important for the establishment of heterochromatin would likewise be defective for heterochromatin assembly if transiently depleted in the tas3WG background. In contrast, genes with a less critical role for heterochromatin assembly might be expected to assemble heterochromatin efficiently if transiently depleted in tas3WG cells. Here, we directly test the contribution of proteins involved in the RNAi pathway or in methylation of H3K9 to the initiation of centromeric heterochromatin. We find that transient depletion of any Clr-C component perturbs the establishment of heterochro- matin in tas3WG cells. In contrast, transient depletion of genes involved in the RNAi pathway does not block heterochromatin assembly in tas3WG cells. Thus there appears to be a continuous requirement for the Clr-C complex, but not RNAi, during heterochromatin assembly in cells bearing a disrupted RITS complex. Consistent with this, RNAi-defective cells retain low levels of the heterochromatin mark, H3K9me2, whereas this mark is completely absent from Clr-C mutant cells. Because RNAi- defective cells maintain residual H3K9me2, it is not heterochro- matin initiation which is being monitored following reintroduction of RNAi components, but more downstream aspects of hetero- chromatin assembly. To determine if RNAi is required for the initial step in heterochromatin assembly, we additionally removed H3K9me2 from RNAi defective cells by making compound mutants with clr4D, and interrogated whether on re-expression of clr4+, Clr-C could target centromeric sequences. We found that Clr4 can promote de novo methylation of centromeric repeats when overexpressed in cells otherwise lacking Clr4 and either of PLoS Genetics | www.plosgenetics.org 2 October 2010 | Volume 6 | Issue 10 | e1001174