Figure 2. Characterization of the H3^D/H3^H strain.

(A) Mononucleosomes prepared from both the cells expressing Myc-tagged H3 (ZL38) and the cells expressing untagged H3 (LHT001) were mixed (Myc-H3 +H3) and immunoprecipitated with the anti-Myc antibody. The same immunoprecipitation (IP) assay was performed with mononucleosomes from strains bearing Myc-H3L130H/H3A110D (Myc-H3^H/H3^D) and Myc-H3A110D/H3L130H (Myc-H3^D/H3^H). The precipitated mononucleosomes were denatured and examined by western blotting with both anti-Myc (Myc) and anti-histone H3 N-terminal antibodies (H3N). (B) Chromatin IP (ChIP) analysis was performed in Myc-H3A110D/H3L130H (Myc-H3^D/H3^H) and Myc-H3L130H/H3A110D (Myc-H3^H/H3^D) cells using anti-Myc antibody. The precipitated DNA was analyzed by qRT-PCR with primers specific for the GAL1-10 gene promoter and normalized to the ACT1 gene. Error bars indicate s.e.m. for three independent experiments. (C) MNase digestion of nuclei from WT (LHT001) and H3^D/H3^H strains. Nuclei were digested with increasing concentrations of MNase for 4 min. MNase cleavage sites were mapped from the EcoRI site within GAL10 by indirect end labeling analysis on a 1.6% agarose gel. Marker fragments are from PCR products of 1 kb and 500 bp in length. The UAS region and nucleosome positions are schematically shown on the right. ND, naked DNA. (D) Dotting assays were performed in H3^D/H3^H mutant and WT (LHT001) cells. Plates were photographed after incubation at 37°C, 30°C and 23°C on yeast extract peptone dextrose (YPD) medium or after incubation at 30°C on YPD, YPD containing phleomycin and YPD containing MMS on Days 1, 2 and 3. (E) Growth curve assays were performed in H3^D/H3^H mutant and WT cells for the indicated time in medium containing different carbon sources. (F) Yeast chromatins extracted from WT (LHT001) and H3^D/H3^H strains were monitored by western blot analysis with antibodies against H3K4ac, H3K4me2, H3K4me3, H3K18ac, H3K27ac, H3K36me3, H3N (H3 N-terminal), H4ac, H4K16ac and H4. Signals are normalized by anti-H4 antibody. (G) Scatter plot showing the average Reads Per Kilobase per Million mapped reads (RPKM) of two replicates distribution of the WT (LHT001) and H3^D/H3^H strains. The Pearson's product-moment correlation of Log₁₀(WT_average_RPKM +1) and Log₁₀(H3^D/H3^H _average_RPKM +1) is 0.9236. The red line is the fitted curve, which has a slope of 0.9966 and which passes through the (0,0) point. R-square is 0.98 and p value≤2.2e-16 (see Materials and methods for details).

Figure 2—figure supplement 1. Mononucleosome preparation from H3^D/H3^H cells.

(A) Mononucleosomes were prepared from H3^D/H3^H cells, and purified by fast protein liquid chromatography. Molecular standards are labeled above the elution profile. (B) Fractions around the peak fractions (No. 14 to 31) were collected and denatured. The length of DNA was analyzed in a 2% agarose gel. Size markers are on the left. (C) Histone H3 in the fractions (labeled on top) was examined by western blotting using the anti-H3 N-terminal antibody. Fraction 26 was selected as mononucleosomes for further experiments.

Figure 2—figure supplement 2. Sample-to-sample reproducibility for the RNA-Seq assay of WT (LHT001) and H3^D/H3^H strains.

(A) The scatter plot shows the FPKM distribution for the two WT replicates. The Pearson's product-moment correlation for WT_rep1 and WT_rep2 is 0.9907. The red line is the fitted curve, which has a slope of 0.8711 and which passes through the (0,0) point. R-square is 0.9823 and p value≤2.2e-16. (B) Scatter plot showing the FPKM distribution for the two H3^D/H3^H's replicates. The Pearson's product-moment correlation for H3^D/H3^H_rep1 and H3^D/H3^H_rep2 is 0.9913. The red line is the fitted curve, which has a slope of 0.9875 and which passes through the (0,0) point. R-square is 0.9846 and p value≤2.2e-16.