(A) Schematic illustration of asymmetrical mutation of histone H3K36R on either H3A110D (H3D) or H3L130H (H3H). The K to R mutation is marked by ® on the histone H3. The color labeling of each strain is applied for all the corresponding panels. (B) Asymmetrical K36me3 on sister H3s is successfully established on chromatin. Nucleosomes were immunoprecipitated by anti-H2B antibody from isogenic strains, as identified by colored numbers, and analyzed by western blot using anti-H3K4me3, anti-H3K36me3 and, as a normalization, anti-H4 antibody. (C) Quantification of western blot signals for H3K36me3 as mean ratio relative to H3D/H3H and normalized to signals for H4. (D) Northern blot analysis of the FLO8, STE11 and PCA1 transcripts in H3K36R mutants. RNA from H3D/H3H, H3DK36R/H3H, H3D/H3HK36R and H3DK36R/H3HK36R strains was probed with sequences complementary to the 3' region of FLO8, STE11, PCA1 and, as a loading control, SCR1. The full-length (FL) and short transcript signals are indicated. (E and F) Asymmetric K36me3 on sister H3s results in an intermediate level of H4ac in FLO8, STE11 and PCA1. ChIP experiments were performed in 3' ORF region of FLO8, STE11 and PCA1 in the indicated cells with anti-H3K36me3 antibody (E) and anti-H4ac antibody (F). Values are normalized to histone H4 and expressed as mean ratio to H3D/H3H. (G) H4ac level of the FLO8, STE11 and PCA1 loci in set2∆ cells bearing different K36me3 states on sister H3s. SET2 was knocked out in the indicated cells and ChIP experiments were performed as in (E). Values are normalized to histone H4 and expressed as mean ratio to H3D/H3H. The H3D/H3H cells are regarded as a WT control. In all cases, the values of H3D/H3Hare set to 1. All error bars indicate s.e.m. for at least duplicated experiments.