Fig 1: Sir2, Clr3 and Swi6HP-1 are required for heterochromatin maintenance in RNAi-compromised cells. (A) Silencing assay at centromere otr1R(SphI):ade6+. Left panel: diagram indicating ade6+ reporter gene insertion at dg of cen1. The position of PCR products (p-dg and p-dh) is also indicated. The primers also hybridize with centromere 2 and 3 (Supplementary Figure S2A). Right panel: serial dilutions of cells were spotted onto limiting adenine medium. In wt cells, ade6+ is repressed (red colonies); silencing-compromised mutants alleviate the repression (pink/white colonies). (B) Serial dilutions of cells were spotted onto non-selective (NS) plates or 10µg/ml TBZ-containing medium. (C) Diagram of procedure to assess the heterochromatin maintenance on minichromosome containing two dg elements (MC-dg'') in wt and mutant cells. R and V indicate regions on minichromosome analysed by qPCR. (D) Colony colour assay to assess minichromosome stability. Cells containing MC-dg'' were crossed into wt and indicated mutants, and plated on limiting adenine plates. Red colonies indicate unstable minichromosomes; white/sectored colonies indicate stable minichromosomes that are retained at mitosis. (E) Percentage of MC-positive white/sectored colonies in indicated host strains. (F) qChIP analyses of H3K9me2 levels maintained on MC-dg'' following crosses into wt and indicated mutant strains. Enrichment is shown relative to actin (act1+), and normalized to wt. R and V correspond to regions on minichromosome analysed by qPCR. (G) qChIP analyses of H3K9me2 levels associated with endogenous centromere dh (top; p-dh) and dg (bottom; p-dg) elements. Enrichment is shown relative to actin (act1+), and normalized to wt. (H) Immunofluorescence analysis of GFP-Swi6 in wt or mutant cells. Representative images show staining of GFP-Swi6 (green), CENP-ACnp1 (red) and DNA (DAPI-blue). (I) qChIP analyses of GFP-Swi6 levels associated with endogenous centromere dg repeats. Enrichment is shown relative to actin (act1+), and normalized to wt. Error bars for (F), (G) and (I): s.d. of three biological replicates.