Clr-C Required for Heterochromatin Establishment Figure 3. Pcu4 is required for establishment of repressive centromeric heterochromatin. A. Real time PCR analysis of centromeric transcripts from dh sequences relative to adh1+ in cDNA derived from the indicated strains. Data represent mean 6 SEM, normalized to wild type strain (PY41). Strains used were PY1065, PY2268, PY41, PY1797, PY3515, PY5082, PY5083, PY5085, PY5086. B. Real time PCR analysis of centromeric transcript accumulation from dg repeats. Samples were processed as described in (A). C. Northern blotting for dh siRNAs and SnoR69 as a loading control in small RNAs from strains listed in (A). D. ChIPs of H3K9me2 (upper panel) and Chp1 (lower panel) association with centromeric dh sequences, with adh1 serving as control. Strains analyzed were as for (A), and data represents mean 6 SEM from duplicate ChIP experiments, quantified by real time PCR. doi:10.1371/journal.pgen.1001174.g003 likely due to defective reassembly of the Clr-C complex following transient depletion of pcu4, since H3K9 methylation resumes effectively in pcu4+ reconstituted tas3-TAP cells. Chp1 binds H3K9me2/3 and Chp1 recruitment to centromeres is a hallmark of heterochromatin. ChIP experiments performed with anti-Chp1 antibodies demonstrated that pcu4D cells are also defective for Chp1 association with centromeres, and pcu4+ reconstitution of pcu4D tas3-TAP but not of pcu4D tas3WG-TAP cells promotes Chp1 association with centromeres (Figure 3D, lower panel). We also assessed H3K9me2 levels at centromeres in other strain backgrounds. In all Clr-C mutants (raf2, rik1, raf1), centromeric H3K9me2 levels were no higher than seen in clr4D cells (Figure 4A and 4C). Following reconstitution with raf2+, clr4+, rik1+, or raf1+, tas3-TAP cells accumulated ‘‘wild type’’ levels of H3K9me2, but tas3WG-TAP cells failed to accumulate H3K9me2 at centromeres. Very similar results were obtained for Chp1 association with centromeres (Figure 4B and 4D), consistent with tas3WG-TAP cells being dependent on constitutive expression of all components of the Clr-C complex to provide H3K9me2 at centromeric sites for recruitment of Chp1. In sum, these experiments demonstrate that in cells where the association of Ago1 with Chp1-Tas3 has been abrogated, that reintroduction of Clr-C components is not sufficient to direct H3K9me2 accumulation on centromeric repeats. Clr-C defective cells should still express heterochromatin independent siRNAs, and Ago1 in these cells would be expected to maintain association with primal RNAs. Thus targeting of Ago1 by priRNAs to centromeric repeats is not sufficient to drive Clr-C recruitment to centromeres when Ago1 is physically separated from Tas3-Chp1. Dicer knockout cells show efficient assembly of centromeric heterochromatin on reintegration of the dcr1+ gene Next we examined whether RNAi components contribute to the initial steps in heterochromatin assembly. RNAi defective cells, such as dcr1D, are expected to retain priRNAs but lose most of their siRNAs. In contrast to Clr-C defective cells, dcr1D cells maintain low levels of H3K9me2 at centromeres (Figure S2D). Following overexpression of dcr1+, both dcr1D tas3-TAP and dcr1D tas3WG -TAP cells efficiently assembled heterochromatin [10]. This suggested that H3K9me2, and not siRNA, acts at an early stage of heterochromatin initiation. However, in these experiments it was unclear whether overexpression of dcr1+ suppressed an establish- ment defect in tas3WG dcr1+ reconstituted cells [10,14]. We directly tested whether integration of dcr1+ into the genomic dcr1D locus of tas3-TAP and tas3WG-TAP cells could support reassembly of centromeric heterochromatin. Cells lacking dcr1+ fail to silence the cen::ura4+ centromeric transgene. Following reinte- gration of dcr1+, silencing of the cen::ura4+ reporter resumed in both dcr1D to dcr1+ tas3-TAP and dcr1D to dcr1+ tas3WG-TAP cells (Figure S2A). Cells lacking dcr1+ accumulate high levels of centromeric transcripts, but following dcr1+ reintegration, centromeric tran- script levels were reduced in both the dcr1+ reconstituted tas3-TAP and tas3WG-TAP cells, confirming that reintegration of dcr1+ promoted efficient assembly of centromeric heterochromatin (Figure S2B, S2C). In addition, dcr1D cells cannot generate siRNAs from centromeric transcripts, but on reintegration of dcr1+, siRNA production resumed efficiently in both tas3-TAP and tas3WG-TAP backgrounds (Figure S2D). Together, these results confirmed and extended our data obtained with overexpressed dcr1+ [10]. dcr1+ and siRNAs are not critical for Clr-C activity at centromeres, but are important for amplification of the H3K9me2 signal during later stages of heterochromatin assembly. Components of the RDRC complex are not required for heterochromatin assembly in tas3WG cells We next asked whether transient depletion of genes that act upstream of Dcr1 in the RNAi pathway would cause defective heterochromatin establishment. RDRC acts upstream of Dcr1, generating dsRNA for siRNA production. RDRC consists of the RNA-dependent RNA polymerase (Rdp1), the RNA helicase Hrr1, and a non-canonical poly (A) polymerase, Cid12 [11]. Cells lacking any component of RDRC show reduced RITS association and reduced H3K9me2 at centromeres, and have reduced siRNA production [11,19,20]. We introduced the tas3-TAP and tas3WG-TAP alleles into deletion mutants of all components of RDRC, and then tested whether silenced chromatin assembled on the cen::ura4+ reporter following reintegration of genomic clones encoding these genes. For cells lacking cid12+, hrr1+, or rdp1+, reintegration of these genes into knockout tas3-TAP cells allowed efficient assembly of heterochromatin. Interestingly, as seen for Dcr1, reintroduction of the genes into the mutant tas3WG-TAP strains also supported silencing of the cen::ura4+ reporter (Figure 5A and 5B, Figure 6A). Transcript analyses performed on the RDRC reconstituted strains revealed that cells lacking RDRC components accumulate both centromeric dg and dh transcripts, but that on reconstitution with the wild type gene, dg and dh transcript levels dropped to levels close to those normally found in tas3-TAP or tas3WG-TAP cells, which is considerably less than seen in RDRC mutant cells (Figure 5C and 5E, Figure 6C, and Figures S3A, S3B, and S4A). Thus processing of centromeric transcripts is efficiently resumed following reintroduction of RDRC components into RDRC deficient tas3WG cells, and this conclusion is further supported by detection of siRNAs in RDRC reconstituted cells (Figure 5D, 5F and Figure 6B). In summary, in contrast to cells transiently depleted for Clr-C components, centromeric heterochromatin assembly can occur efficiently following the transient depletion of RDRC components or of dcr1+ in tas3WG cells. Low levels of H3K9me2 at centromeres in RDRC deficient cells support assembly of heterochromatin in reconstituted tas3WG cells Cells lacking dcr1+ accumulate H3K9me2 on centromeric sequences, whereas Clr-C deficient cells completely lack H3K9me2. We therefore asked whether cells lacking RDRC components accumulate centromeric H3K9me2, and whether H3K9me2 levels could signal the difference in outcome, promoting heterochromatin assembly in tas3WG cells following transient depletion of RDRC, but not Clr-C components. PLoS Genetics | www.plosgenetics.org 7 October 2010 | Volume 6 | Issue 10 | e1001174