Clr-C Required for Heterochromatin Establishment Figure 9. Table summarizing results from this study. Tas3WG cells lacking Clr-C components lack H3K9me, but retain heterochromatin independent siRNAs and priRNAs. These cells cannot support recruitment of Clr-C to centromeres following reintegration of the missing Clr-C component. In contrast, Tas3WG cells lacking RNAi components retain residual H3K9me, but lack siRNAs. On reintegration of the missing RNAi component, these cells convert to allow full heterochromatin assembly on centromeric repeats. These results suggest that Clr-C can function independently of the RNAi pathway in the assembly of centromeric heterochromatin. To further test this hypothesis, H3K9me was withdrawn from RNAi deficient cells to determine whether Clr-C can target centromeres de novo independently of the RNAi pathway. Overexpression of Clr4 in clr4Dago1D cells supports recruitment of Clr-C activity to centromeric repeats to allow initiation of heterochromatin. These data suggest that normally RNAi-independent and RNAi-dependent mechanisms cooperate for full heterochromatin assembly. doi:10.1371/journal.pgen.1001174.g009 activities. This experiment revealed that indeed Clr4, when overexpressed, can initiate H3K9me2 deposition at centromeres independent of Ago1 (Figure 8C). Taken together, our data demonstrate (1) that Clr-C compo- nents can act independently of members of the RNAi pathway to initiate heterochromatin assembly and (2) the ability to promote heterochromatin assembly in tas3WG cells correlates with the prior levels of centromeric H3K9me2 in mutant backgrounds, and not the initial small RNA abundance and (3), that Ago1-bound priRNAs are unlikely to be the key initiator of heterochromatin assembly. The functional data that we present therefore counters the model for heterochromatin initiation proposed recently [20], and supports that RNAi-independent factors, together with the RNAi pathway, are necessary for full heterochromatin assembly. The conclusions that derived from our observations contrast with the widely held belief that RNAi initiates heterochromatin assembly at fission yeast centromeres. Although it has been shown by several labs that small RNAs derived from exogenous hairpin RNAs can induce silencing of genomic loci [37,38], these effects tend to be very weak and very locus specific. In these experiments, silencing efficiency correlates with proximity to sites of hetero- chromatin, or is enhanced by overexpression of heterochromatin proteins. The production of the majority of centromeric small RNAs depends on the presence of heterochromatin. However, low levels of small RNAs are found in Clr-C deletion backgrounds [12,20] or in histone H3K9R mutant cells [19]. Interestingly, Clr-C mutants that completely lack H3K9me are deficient for heterochromatin establishment in our reintegration assay, in spite of the presence of centromeric siRNAs (which are below the level of detection of our Northern assay). In contrast, RNAi-defective strains that are devoid of, or express even lower levels of centromeric small RNAs than Clr-C mutants [12,19,20], can assemble heterochromatin effectively following reintegration of the wild type gene into the tas3WG background. Although not easy to detect, priRNAs, which have been postulated to prime heterochromatin establishment, are expected to be present in all of the genetic backgrounds that we tested for heterochromatin initiation. This class of small RNAs therefore does not appear to contribute to the differential ability of mutants to initiate heterochromatin assembly in the tas3WG background. Finally, our data showing that transient depletion of ago1+ does not impair heterochromatin establishment in tas3WG cells, and that ago1D cells retain H3K9me2, would argue that ago1+ and priRNAs are not the initiating trigger for heterochromatin assembly. These results beg the question of how low levels of siRNA synthesis occur in the absence of heterochromatin, since early models suggested that localization of the RITS and RDRC complexes to centromeres was a prerequisite for centromeric siRNA generation, and that RITS and RDRC complex localization was dependent on Clr4 [8,11]. One study suggested that single-stranded transcripts from centromeric sequences can adopt secondary structures to yield dsRNA that can be targeted by Dcr1 to form siRNAs [19]. Such models for the heterochromatin- independent synthesis of centromeric siRNAs may now help to explain our previously puzzling result that Chp1 chromodomain mutants that are defective for heterochromatin establishment following transient depletion of clr4+ express abundant siRNAs [6]. How low levels of H3K9me2 are initially placed at centromeres remains an open question, but our data supports that it is not absolutely dependent on small RNAs and the RNAi pathway. We suggest that H3K9me2 deposition is linked to the transcription of the centromere. Mutants in three separate components of the RNA pol II complex show defects in heterochromatin assembly, including a mutant that truncates the C terminal repetitive tail of the largest subunit of the polymerase, Rpb1 [16–18]. This raises PLoS Genetics | www.plosgenetics.org 14 October 2010 | Volume 6 | Issue 10 | e1001174