Clr-C Required for Heterochromatin Establishment Figure 7. Ago1 reintegration into tas3WG cells promotes heterochromatin assembly, and ago1 cells retain significant centromeric H3K9me2. A. Real time PCR analysis of cDNA derived from the indicated strains, measuring centromeric dh transcripts normalized to adh1+, and to a wild type strain (PY 42). Data represents the mean 6 SEM from analysis of 2 independent cDNA preparations from duplicate biological samples. Strains used: PY 42, 1798, 1064, 2267, 901, 5193, 5194, 5202, 5203. See also Figure S5A. B. Small RNA Northern blot to identify centromeric dh siRNAs, with SnoR69 as a loading control. Samples used as in (A). C. ChIP for H3K9me2 on centromeric dh repeats, assessed by real time PCR and normalized to adh1 association. Data represents mean of duplicate independent ChIP experiments 6 SEM. Strains as listed in (A). D. ChIP for H3K9me2 on centromeric dh repeats, assessed by real time, and normalized to both act1+ and adh1+ association. Data represent mean of 4 independent ChIP experiments 6 SEM. Strains analyzed are PY42, 1798, 1478, 1550, 901, 5236. Two tailed P value for ago1D vs clr4D and for ago1D vs ago1Dclr4D = ,0.0001 in t test for data normalized to adh1+, and p = 0.0053 (ago1D vs clr4D) and p = 0.0058 (ago1D vs ago1Dclr4D) for data normalized to act1+. E. ChIP for H3K9me2 on centromeric dg repeats, as described for (D). Two tailed P value for ago1D vs clr4D = 0.0005 and for ago1D vs ago1Dclr4D = ,0.0008 in t test for adh1 normalized data, and p = 0.001 (ago1D vs clr4D) and p = 0.0016 (ago1Dvs ago1Dclr4D) for act1 normalized data. doi:10.1371/journal.pgen.1001174.g007 prior H3K9me. To accomplish this, we generated clr4D dcr1D cells and found that some deposition of H3K9me2 at centromeric sequences occurred upon overexpression of clr4+ (Figure 8C). These experiments clearly demonstrate that Clr-C can function to initiate de novo centromeric heterochromatin assembly indepen- dently of the RNAi pathway. Recently, Ago1 and its associated priRNAs have been proposed to trigger heterochromatin formation. This notion is partly based on an observation that an ago1D strain had little or no H3K9me2 at centromeres, suggesting an upstream role for Ago1 [20]. If this hypothesis were correct, we anticipated that in our tas3WG system, that transient depletion of Ago1 might block heterochromatin assembly similar to what we observed for Clr4. In contrast, we found that tas3WG cells formed robust centromeric heterochroma- tin on reintroduction of ago1+ into ago1D cells (Figure 7A–7C). Consequently, we re-examined the reported critical Dicer- independent role for Ago1 in driving H3K9Me. In contrast to a recent study [20], we found that H3K9me2 levels in ago1D cells were no lower than in other RNAi-defective backgrounds (Figure 7D and 7E). To further probe for a potential role of priRNAs in heterochromatin initiation, we generated cells that lack both Ago1 (which binds priRNAs) and Clr4, and tested whether reintroduction of Clr4 could promote de novo centro- meric H3K9me2 in the absence of Ago1-priRNA targeting Figure 8. Initial Clr-C recruitment to centromeres can occur independently of ago1+ and dcr1+. A. Schematic for removal of residual H3K9me2 in dcr1D or ago1D cells by introduction of clr4D, prior to re-expression of genomic clr4+ from episomal vector to test for Clr-C’s ability to promote de novo heterochromatin assembly in RNAi-deficient tas3WG cells. B. Removal of residual H3K9me2 in dcr1D and ago1D cells was performed by generation of double mutants with clr4D, and then ability of Clr-C to generate de novo centromeric H3K9me2 methylation was tested in absence of RNAi pathway following transformation with genomic clr4+ plasmid. H3K9me2 ChIP was performed on indicated strains, and assessed by real time PCR analysis. Strains analyzed: PY12, 5516, 5557, 5517, 1637, 5522, 5523, 5520, 5521. All were grown in PMG-his media. Data represent mean of 3 or 4 ChIPs 6SEM for all samples except dcr1D and WT for which 1 ChIP is shown. doi:10.1371/journal.pgen.1001174.g008 PLoS Genetics | www.plosgenetics.org 13 October 2010 | Volume 6 | Issue 10 | e1001174