Supplement to Peng et al., 2008

Supplemental Tables and Figures

Files in this Data Supplement:

• The nuclear localization of Gcn5 was not affected in the indicated deletion mutants. - "Gcn5 in the indicated mutants was myc-tagged in its chromosomal locus at the 3¡¦ end. The monoclonal anti-myc antibody (9E10) (Roche, IN) and FITC-conjugated secondary antibody were used to detect the localization of Gcn5 by indirect immunofluorescence. Wild-type and gcn5ƒ´ strains were used as controls."
• Expression of HAT and HDAC complex components. - "Changes in expression of histone modifiers are unlikely to be the reason for reduced cellular levels of histone acetylation in vma5£G, vma8£G and vps45£G (Lib) mutants. Most HATs, HDACs and their associated components in vma5£G (A, B and C), vma8£G (D, E and F) and vps45£G (Lib) (G, H and I) are expressed at nearly normal levels as compared to the wild type strain."
• Comparison of growth rates of the identified deletion mutants with low global histone acetylation levels. - "Overnight cultures (YPD) of the indicated strains were adjusted to a density OD600 of 1.5 and spotted on YPD plates at 10-fold serial dilutions. Pictures were taken after 48 hr-incubation at 30¢X."
• Histone H3K79 methylation levels in the identified deletion mutants. - "Shown are the percentages of cellular levels of unmethylated and mono-, di-, and tri-methylated H3K79 in each indicated strain as determined by mass spectrometry."
• The vps45 deletion from the single gene deletion library may harbor additional mutations. - "(A) The levels of histone H3 acetylation in wild type, vps45£G(Lib) and vps45£G (Lab) (CTY135) are determined by Western blotting with an anti-H3K9/K14ac antibody. The library strain, vps45£G(Lib), show much lower levels of H3 acetylation compared to the one generated by our laboratory (vps45£G (Lab)). However, both deletion strains show lower levels of acetylation compared to wild type. (B) The Gcn5-myc protein was immunoprecipitated from wild type, ada2£G and vps45£G (Lib) cells and subjected to 4-20% gradient Tris-HCl gel (Bio-Rad, CA) electrophoresis and silver staining. As indicated by the arrows, certain Gcn5 associated factors are missing or modified in the vps45£G (Lib) strain which may explain the significant reduction in H3 acetylation and Gcn5 HAT activity."
• Reduction in cellular levels of histone acetylation maps to specific genomic loci. - "Levels of histone acetylation at (A) H3K18 and (B) H3K9/K14 were determined at open reading frame (o) regions of PYK1, MEC1 and PMA1 genes by standard chromatin immunoprecipitation (ChIP). A region 500 bp from telomere of chromosome VI-R (TEL) was used as internal control in each PCR reaction. The values reflect enrichment of ChIPed DNA relative to TEL region and input DNA and shown as percentage of wild type which was set to 100%. The data are averages of three independent experiments."
• Percentage of total histones that are acetylated at indicated residues in histones H3 and H4. - "Supplemental Table 1"
• The cellular decrease in histone acetylation in the deletion mutants can be rescued by reintroduction of the wild type copy of the gene. - "The indicated deletion mutants were complemented with a wild type copy of the deleted gene on the BG1805 vector (Open Biosystems) under the control of the GAL1 promoter. URA3 auxotrophy was used for selection. Histones were extracted from the indicated strains after 6 hr growth in repressible (SD-URA) or inducible (SG-URA) conditions, and were subjected to standard Western blotting with anti-H3 and anti-H3K9/K14ac to detect total histone H3 and its acetylation levels, respectively."
• Supplemental Figure Legends - "Supplemental Figure Legends"