We assessed centromeric H3K9me2 levels in RDRC deficient cells and following reintegration of RDRC components. In these experiments, we note that in all RDRC mutants, the level of H3K9me2 at centromeres is considerably higher than seen in cells lacking clr4+, although at least 2 fold reduced compared with wild type cells. Reintegration of RDRC components into the corresponding RDRC cells supported centromeric accumu- lation of H3K9me2 of both tas3-TAP and tas3WG-TAP cells to levels found normally (Figure 6D and 6E). In addition, although Chp1 association with centromeres is diminished in hrr1D cells, reintroduction of hrr1+ into tas3WG-TAP cells promoted Chp1 association (Figure S4B). Together then this data shows that heterochromatin assembly occurs efficiently following transient depletion of genes required for siRNA synthesis, including RDRC components that act upstream of Dcr1. In addition, the ability of heterochromatin to reform efficiently, following transient deple- tion of RNAi components in tas3WG cells, correlates with the persistence of low levels of H3K9me2 on centromeric repeats in the mutant backgrounds. Transient depletion of Ago1 in Tas3WG cells causes no defect in heterochromatin establishment Very recently it has been proposed that Ago1 is the most upstream factor in heterochromatin assembly. It is thought to act as an acceptor for RNA degradation products, termed pri-RNAs, which, based on frequency of occurrence, preferentially target antisense transcripts resulting from bidirectional transcription of DNA repeats. Cleavage of nascent centromeric transcripts by priRNA-directed activity of Ago1 is proposed to recruit the RDRC complex and eventually promote siRNA-dependent recruitment of RITS, and subsequent robust assembly of heterochromatin via recruitment of Clr-C [20]. This model therefore places Ago1 as an initiator, upstream of RDRC and Dcr1 and of the Clr-C complex and H3 K9 methylation. This model is supported by the detection of small RNAs (priRNAs) in dcr1D strains, and of siRNAs in cells lacking clr4+, or in which heterochromatin assembly is blocked because of mutation of H3K9, supporting that heterochromatin is not essential for small RNA generation [19,20]. Finally, the model would suggest that siRNAs and priRNAs act upstream of heterochromatin assembly, and that Ago1 is the most upstream component of the RNAi pathway. Indeed, Halic and Moazed argue that Ago1 activity is required for the initial deposition of H3K9me, since in their publication strains lacking Ago1 exhibit lower levels of centro- meric H3K9me accumulation than strains deficient in other components of the RNAi pathway [20]. To test the role of Ago1 in heterochromatin assembly, we first performed transient depletion experiments for Ago1 in the tas3WG- TAP background (Figure 7). We integrated a genomic clone of ago1+ into ago1 tas3-TAP and tas3WG-TAP cells, and monitored heterochromatin assembly. In contrast to ago1 cells, where centromeric transcripts are highly elevated (above the levels seen in clr4D cells), reintroduction of ago1+ into either tas3-TAP or tas3WG-TAP cells reduced centromeric dh and dg transcripts to levels seen normally in tas3-TAP and tas3WG-TAP cells (Figure 7A and Figure S5A). Consistent with this suppression, we found that unlike ago1 cells, where siRNA production is severely reduced, centromeric siRNAs are synthesized at normal levels following reintroduction of ago1+ (Figure 7B). Next, we performed ChIP experiments to monitor H3K9me2 levels in ago1D cells. ago1 deletion reduces H3K9me2 accumulation at centromeres below that of wild type cells, but above that of clr4D cells. On reintroduction of ago1+, centromeric H3K9me2 levels accumulate to normal levels (Figure 7C), similar to the results seen on Clr-C Required for Heterochromatin Establishment reintegration of other RNAi components into tas3WG cells. These data suggest that cells lacking ago1 behave similarly to other RNAi defective strains, and functionally that there is sufficient H3K9me2 in ago1D tas3WG cells to drive heterochromatin assembly following reintroduction of ago1+. We further analyzed centromeric H3K9me2 levels in RNAi defective strains. At the 2 sites tested within centromeric dg and dh repeats, H3K9me2 levels were significantly elevated in ago1D cells above the background levels in clr4D or ago1D clr4D cells, and H3K9me2 accumulation at centromeres was similar in all RNAi deficient backgrounds tested. This data would suggest that Ago1, like other RNAi components, is not acting upstream of Clr-C for heterochromatin assembly. RNAi pathway is not required for initial step in heterochromatin initiation Our demonstration that heterochromatin can assemble follow- ing transient depletion of RNAi components in tas3WG cells is suggestive that the RNAi pathway is acting downstream of Clr-C. However, given that low levels of centromeric H3K9me2 are maintained in RNAi-defective cells, it is difficult to assess whether RNAi is required for the initial step in heterochromatin initiation. To address this question, we removed residual H3K9me2 from RNAi defective cells by introduction of the clr4D allele. We then tested whether H3K9me2 could be deposited at centromeres following expression of Clr4 in these cells that lack both Clr4 and Ago1 or Clr4 and Dcr1 (Figure 8A). Following overexpression of clr4+ in ago1Dclr4D cells, H3K9me2 could be detected on centromeric repeats above the background observed in clr4 cells, and similar to levels found normally in ago1D cells. Similar results were obtained following overexpression of clr4+ in dcr1Dclr4D cells (Figure 8B). Thus, when overexpressed, Clr4 can target centromeric repeats to initiate H3K9me2 deposition in the absence of a functional RNAi pathway. We note, however, that reintroduction of clr4+ into its normal locus in these cells is not sufficient, in the absence of the RNAi pathway, for accumulation of detectable centromeric H3K9me2 (data not shown). Together, these experiments strongly indicate that Clr-C can initiate H3K9me deposition at centromeres via RNAi-independent mechanisms, but that cooperation between RNAi-dependent and RNAi-independent factors normally results in full hetero- chromatin assembly (summarized in Figure 9). Discussion We have utilized our novel mutant, tas3WG, to identify genetic requirements for heterochromatin initiation as opposed to those required for the maintenance of pre-existing heterochromatin. We used an approach in which genes required for heterochromatin formation are deleted and reintroduced and then heterochromatin assembly is examined. In wild-type cells, heterochromatin can be established regardless of the factor removed, indicating that establishment mechanisms are robust to perturbations of the system. However, in cells harboring a disrupted RITS complex, we found that the establishment of silencing becomes sensitive to the prior presence of particular silencing factors. Our data demonstrate that the ability to assemble heterochromatin in such gene removal-restoration experiments in tas3WG cells correlates with the prior presence of H3K9me2 on centromeric repeats, but does not require the prior presence of small RNA species. These data strongly suggest that RNAi-independent mechanisms of recruitment of Clr-C play a key role in the assembly of centromeric heterochromatin. We also tested whether RNAi is required in an obligate manner to initiate de novo heterochromatin assembly in cells that lack any PLoS Genetics | www.plosgenetics.org 11 October 2010 | Volume 6 | Issue 10 | e1001174