Extended Data Fig. 3: The siRNA driver locus is critical for establishing a heritable silent epiallele.

a, cen::ade6⁺ leo1∆ (top) or ade6⁺ hairpin (HP) leo1∆ (bottom) cells were plated on low adenine medium. Approximately 12% of cen::ade6⁺ leo1∆ (top) and 100% of ade6⁺ HP leo1∆ (bottom) cells formed red or pink colonies, indicating silencing of endogenous ade6⁺. Experiment repeated twice. b, c, cen::ade6⁺ ade6⁺ OFF leo1∆ cells (b) or ade6⁺ HP ade6⁺ OFF leo1∆ (c) cells were crossed to cen⁺ ade6^BC+ ON cells and RSA was performed. Number of progeny matching each indicated genotype and phenotype is shown. 80 red and 80 white colonies were genotyped using PCR. Results reflect two independent crosses. d, siRNA sequencing showing limited secondary siRNA generation in ade6⁺ HP leo1∆ ade6⁺ OFF cells, compared with more extensive secondary siRNA spreading to neighbouring genes bub1⁺ and vtc4⁺ in cen::ade6⁺ leo1∆ ade6⁺ OFF cells. Shaded areas represent sequence identity to ade6⁺ HP (top three rows) or cen::ade6⁺ (bottom three rows). Two independent clones shown for each experimental sample. e, f, H3K9me2 (e) and H3K9me3 (f) ChIP–seq reads mapping to the endogenous ade6⁺ locus in cells with the indicated genotypes and expression states. Shaded areas represent sequence identity to ade6⁺ HP (top three rows) or cen::ade6⁺ (bottom three rows). Two independent clones are shown for each experimental sample. g, h, ChIP–qPCR assays showing differences in H3K9me2 (g) or H3K9me3 (h) levels in cen::ade6⁺ leo1∆ ade6⁺ OFF and ade6⁺ HP leo1∆ ade6⁺ OFF cells at ade6⁺ (purple) and vtc4⁺ (blue). Data are mean ± s.d. from three biological replicates. P values are based on two-tailed Student's t-test comparing leo1∆ cells to appropriate wild-type cells. On the right, control ChIP–qPCR at dg repeats.