Clr-C Required for Heterochromatin Establishment represented. B. Serial dilution assay of yeast bearing the cen::ura4+ marker plated onto complete synthetic media (PMG complete), media lacking uracil (2URA), or complete media supplemented with 5-fluoro-orotic acid (+FOA). In the last 4 rows, the rik1+ gene has been reintegrated into the rik1D locus (rik1D to rik1+). Strains analyzed: PY2036, 3776, 3778, 4879, 4880, 4881, 4882. C. Real time PCR analysis of centromeric transcripts from the dh repeats (site A), relative to the adh1+ euchromatic control in cDNA from indicated strains. Duplicate experiments used independent RNA samples, and data represents the mean 6 SEM. All values were normalized to the value obtained for a wild type cen::ura4+ strain (PY2036). Strains used: PY 2036, 1838, 3494, 3497, 3776, 4879, 4880, 4881, 4882. D. Real time PCR analysis performed as described for (C), but measuring the transcript accumulation from the dg repeats (site B) relative to adh1+. E. Northern blot of small RNA populations isolated from indicated strains, hybridized with probes for siRNAs derived from the dh repeat, or SnoR69 RNA as loading control. Strains used as in (C). doi:10.1371/journal.pgen.1001174.g001 cells which lack H3K9me2 (Figure 3D, upper panel). Following pcu4+ reintroduction into pcu4D tas3-TAP cells, H3K9me2 levels rise to that seen in tas3-TAP cells, whereas no significant accumulation of H3K9me2 is observed in pcu4+ reconstituted tas3WG-TAP cells. Thus Clr4 mediated H3K9 methylation is abrogated in tas3WG-TAP cells transiently depleted for pcu4. This methylation defect is not Figure 2. Clr-C components raf1+ and raf2+ facilitate establishment of repressive centromeric heterochromatin. A. Serial dilution assay of raf1+ reintegration strains bearing the cen::ura4+ reporter, and plated on specified media. Strains analyzed: PY2036, 3287, 3659, 3707, 3708, 3710, 3711. B. Serial dilution assay to monitor heterochromatin establishment in raf2D cells into which raf2+ has been reintegrated in tas3-TAP and tas3WG- TAP backgrounds. Strains analyzed: PY2036, 3675, 3676, 3781, 3783, 3791, 3792. C. Real time PCR analysis of dh centromeric transcripts relative to transcripts from adh1+. Data shown represent the mean 6 SEM measurements from cDNA derived from two independent cultures, normalized to the wild type cen::ura4+ strain (PY2036). Strains analyzed: PY2036, 3287, 3659, 3707, 3710, 3711. See also Figure S1A. D. Northern blotting for dg and dh siRNAs and for the loading control SnoR69 on small RNA populations derived from indicated strains (as used in (C) and PY3708). E. Real time PCR analysis of cDNA to monitor centromeric transcript accumulation following reintegration of raf2+ into raf2D cells bearing the tas3-TAP and tas3WG-TAP alleles. Transcripts from dh were measured relative to the adh1+ control. Data were normalized to wild type cen::ura4+ strains, and represents mean 6 SEM. Strains analyzed: PY2036, 3494, 3497, 3293, 3781, 3783, 3791, 3792. See also Figure S1B. F. Northern blot analysis of siRNAs derived from dg and dh regions of the centromere, with SnoR69 as a loading control. Strains were as listed in (E). doi:10.1371/journal.pgen.1001174.g002 PLoS Genetics | www.plosgenetics.org 5 October 2010 | Volume 6 | Issue 10 | e1001174