(A) Displayed is the MS/MS fragmentation spectrum of the [M+2H]²⁺ parent ion from an H3 peptide (histone H3, amino acids 29–37) derived from a propionylated digest of Tetrahymena H3. Inset shows full MS of parent ion (whole peptide), from which the fragmentation spectrum was taken, at 542.3062 m/z. Scale for the y-axis of inset represents relative abundance of the parent ion and is identical to the fragmentation spectrum y-axis. Accurate mass (inset) indicates acetylation and not trimethylation on this peptide (+0.18 ppm error), while fragment ions (full spectrum) show H3K36 as the acetylation site. Experimentally observed (MH²⁺ _exp.) and calculated masses (MH²⁺ _calc.) for this acetylated peptide are indicated. Above the spectrum is the peptide sequence in which predicted b-type ions, which contain the amino terminus of the peptide, are immediately above the sequence. Predicted y-type ions, which contain the carboxyl terminus, are immediately below the sequence. Ions observed in the spectrum are underlined and represent masses associated with the fragmented peptides from the MS/MS analyses. Note the addition of propionyl groups (Pr, adds 56 Da) on amino terminus residues and unmodified lysine residues due to chemical derivatization with propionic anyhydride reagent (see experimental procedures for explanation). (B) Same as in (A) except peptides were derived from digested RP-HPLC purified yeast H3. Displayed is the MS/MS fragmentation spectrum of the [M+2H]²⁺ parent ion at m/z 823.4552. This peptide was identified as the 27–40 fragment from yeast histone H3. Inset shows full MS of parent ion at m/z 823.4552. Accurate mass indicates the addition of two acetylation modifications and not trimethylation on this peptide (+0.73 ppm error), while fragment ions show H3K27 and H3K36 as the acetylation sites. Experimentally observed (MH²⁺ _exp.) and calculated masses (MH²⁺ _calc.) for this acetylated peptide are indicated. As in (A), b- and y-type ions observed in the spectrum are underlined and the peptide contains the addition of propionyl groups (Pr) on unmodified lysine and amino terminus residues.

(A) An antibody specific to H3K36 acetylation recognizes Tetrahymena H3. RP-HPLC Tetrahymena H3 (same as used in Fig. 1A) was loaded onto adjacent lanes and resolved on a 15% SDS-PAGE gel. Following transfer to a polyvinylidene difluoride (PVDF) membrane, each lane was separated and probed with an α-H3K36 acetyl antiserum (α-H3K36ac) that was preincubated with different unmodified or modified H3 synthetic peptides as indicated. The same blots were stripped and reprobed with an antibody specific for Tetrahymena H3 (α-Tetrahymena H3) as a loading control. (B) The α-H3K36ac antibody specifically recognizes H3K36 acetylation in yeast. Acid-extracted histones prepared from a wild-type or H3K36 point mutant yeast strain (K36A) were resolved on a 15% SDS-PAGE gel, transferred to a PVDF membrane, and probed for H3K36ac. An antibody specific for the C-terminus of H3 (α-H3) was used as a loading control. Antibodies specific for H3K18 acetylation (α-H3K18ac) and H3K36 trimethylation (α-H3K36me3) were used as additional controls.

(A) The distribution of average z-scores (units are standard deviation from the mean) for 5′ regulatory regions (black), ORFs (red), and 3' UTRs (blue) derived from ChIP-chip experiments in which H3K36ac ChIPs were compared directly to H3K36me2 ChIPs. Thus, the H3K36ac and H3K36me2 ratios shown here are inversely related. Similar promoter enrichment results for H3K36ac were obtained when the H3K36ac ChIPs were compared to a genomic DNA reference, or references composed of histone H3 ChIPs (experimental procedures). (B) A moving-average plot (window size=40, step size=1) of average z-scores from three independent experiments comparing H3K36ac ChIPs (red), H3K9/14ac ChIPs (black) and H3K36me2 ChIPs (blue) on a high resolution DNA microarray covering all of chromosome III. ChIP enrichment is plotted as a function of the distance from the translational start site among genes greater than 1 kb in length. (C) H3K36ac distribution genome-wide. Colors (scale at bottom) represent the median of all z-scores recorded from all arrayed elements in the indicated functional class (labeled on right, number of elements indicated in parentheses). Data were derived from three independent replicates.

(A) Shown are the results of a HAT assay in which TAP-purified SAGA complex was incubated with either unmodified or modified H3 synthetic peptides along with unlabeled acetyl coenzyme A (acetyl-CoA). Reaction products were resolved on a 10% SDS-PAGE gel, transferred to a PVDF membrane, and analyzed by immunoblot for H3K36ac. An H3 synthetic peptide acetylated at H3K36 was used as a control for antibody detection. Parallel reactions were performed and examined by Coomassie staining to monitor loading (lower panel). (B) Displayed is a graph representing the results of a HAT assay in which TAP-purified SAGA complex was incubated with either unmodified or modified H3 synthetic peptides along with [³H] labeled acetyl-CoA. ³H incorporation was analyzed by filter-binding assay and monitored by scintillation counting. (C) Shown are the results of a HAT assay in which TAP-purified SAGA complex was incubated with either chicken core histones or recombinant mononucleosomes along with unlabeled acetyl-CoA as in (A). Reaction products were resolved on a 15% SDS-PAGE gel, transferred to PVDF membrane, and analyzed by immunoblot for H3K36ac. Parallel reactions were performed and analyzed by immunoblot for H3K14ac as a control. The same blot was stripped and reprobed with an antibody specific for the C-terminus of H3 (α-H3) to monitor loading. Note slight antibody detection of histone H3 backbone in the absence of TAP-purified SAGA in the recombinant (unmodified) mononucleosomes reactions. (D) Gcn5 is responsible for mediating H3K36ac in yeast. Acid-extracted histones prepared from wild-type, gcn5 Δ or sas3Δ strains were resolved on a 15% SDS-PAGE gel, followed by transfer to a PVDF membrane, and analyzed by immunoblot for the presence of H3K36ac. An antibody specific for the C-terminus of H3 (α-H3) was used as a loading control. Antibodies specific for H3K18ac and H3K36me3 were used as additional controls.

(A) Acid-extracted histones from Tetrahymena, yeast (S. cerevisiae), mouse (mouse embryonic fibroblasts) and human (HEK293) cells, along with recombinant H3 from Xenopus, were resolved on a 15% SDS-PAGE gel, transferred to a PVDF membrane and probed for H3K36ac (upper panel). Prior to immunoblot analysis, the membrane was Ponceau S stained to confirm equal loading of protein (lower panel). (B) Alignment of histone H3 protein sequences (amino acids 30–45) from different eukaryotic species. Divergent residues are highlighted in gray boxes. Asterisk indicates the position of lysine 36 in the H3 sequence.