Extended Data Fig. 7: Increased Raf1 abundance alleviates heterochromatin propagation defects in various mutants.

a, Heterochromatic silencing upon Raf1 overexpression (raf1-oe; Pnmt1-raf1) or loss of upf1 in hht2^G13D (H3.2^G13D) mutant cells. Serial dilution assessed mat2P::ura4⁺ expression, and iodine staining assessed haploid meiosis. b, c, ChIP-seq analysis of H3K14ub (b) and H3K9me3 (c) distribution across the silent mat region in the indicated strains. Note that the WT H3K14ub data is the same as in Fig. 4b as the experiments were performed at the same time. d, Schematic illustrating restoration of heterochromatin propagation upon Raf1 overexpression or loss of Upf1 in hht2^G13D cells. Heterochromatin initiated at the nucleation site through recruitment of ClrC spreads across an extended domain through the Clr4^SUV39H read-write activity, requiring a critical density of H3K9me3 as well as H3K14ub (top). Incorporation of hht2^G13D causes reduction not only in H3K9me3 density, but also H3K14ub, thus disrupting heterochromatin propagation (middle). Elevated Raf1 levels enhance chromatin association of ClrC, H3K14ub and H3K9me3 density, reestablishing heterochromatin spreading (bottom). e, Elevating Raf1 expression in raf1-oe or upf1Δ cells bypass the requirement of other factors for heterochromatin propagation. Serial dilution measured mat2P::ura4⁺ expression, and iodine staining assessed haploid meiosis. Data are representative of two independent experiments. Source data are provided.

Source Data