Insights into the role of histone H3 and histone H4 core modifiable residues in Saccharomyces cerevisiae

Abstract

The biological significance of recently described modifiable residues in the globular core of the bovine nucleosome remains elusive. We have mapped these modification sites onto the Saccharomyces cerevisiae histones and used a genetic approach to probe their potential roles both in heterochromatic regions of the genome and in the DNA repair response. By mutating these residues to mimic their modified and unmodified states, we have generated a total of 39 alleles affecting 14 residues in histones H3 and H4. Remarkably, despite the apparent evolutionary pressure to conserve these near-invariant histone amino acid sequences, the vast majority of mutant alleles are viable. However, a subset of these variant proteins elicit an effect on transcriptional silencing both at the ribosomal DNA locus and at telomeres, suggesting that posttranslational modification(s) at these sites regulates formation and/or maintenance of heterochromatin. Furthermore, we provide direct mass spectrometry evidence for the existence of histone H3 K56 acetylation in yeast. We also show that substitutions at histone H4 K91, K59, S47, and R92 and histone H3 K56 and K115 lead to hypersensitivity to DNA-damaging agents, linking the significance of the chemical identity of these modifiable residues to DNA metabolism. Finally, we allude to the possible molecular mechanisms underlying the effects of these modifications.

FIG. 1.

(A) Sequence alignment of histones H3 and H4 from Saccharomyces cerevisiae and Bos taurus, generated using CLUSTALW. Shaded residues represent the N-terminal tails of the histone proteins. Modified residues identified in bovine histones are labeled and highlighted in both sequences according to the type of modification. (B) Mapping of modifiable residues on the surface of the yeast nucleosome crystal structure (50). A surface representation of the nucleosome, without histone tails, is shown and is viewed down the DNA superhelical axis. Potentially acetylated residues are green, methylated residues are blue, and phosphorylated residues are orange. The DNA helix is represented in bright green. (C) Rotation of view in panel B 90° around the horizontal axis. This is defined as the lateral surface of the nucleosome.

FIG. 2.

(A) Plasmid shuffle experiment of mutant histones to determine their viability. Strains expressing aberrant histones in the presence of wild-type proteins were plated with an initial OD₆₀₀ of 0.5 and serially diluted fivefold on both SC-Trp-Lys and on plates containing α-amino-adipate, which selects against the wild-type histone gene present on the LYS2 “shuffle” plasmid. As a negative control, the JPY12 yeast strain was transformed with a plasmid from which either histone H3 or H4 was completely deleted. (B) Southern blot analysis to determine whether cells expressing the mutated histones were compensating for their presence by increasing the copy number of the plasmid harboring the altered histone. Genomic DNA was extracted from each strain and probed for ACT1, a single-copy gene, and the plasmid backbone in a Southern blot analysis. The ratio of these band intensities was then compared with that of cells containing wild-type histones.

FIG. 3.

(A) Telomeric silencing assay of histone H4 K16, K77, and K79 substitutions. Strains containing an ADE2 reporter inserted into the telomeric regions of chromosome V were transformed with plasmids containing the substituted histones and plated onto SC-Trp plates. After incubation at 30°C for 2 days, plates were put at 4°C for a further 7 days to facilitate the development of the red color. WT and loss-of-telomeric-silencing (LTS) strains are shown for comparisons. (B) ribosomal DNA (denoted rDNA in the figure) silencing assay of histone H4 K77 and K79 substitutions utilizing a strain containing mURA3 and a MET15 marker inserted into ribosomal DNA repeats. Cells were plated with an initial OD₆₀₀ of 0.5 and serially diluted fivefold on SC-Trp for a growth control and SC-Trp-His plus 5-FOA to analyze silencing of the mURA3 reporter. Additionally, colonies were streaked onto complete medium containing PbNO₃ and incubated at 30°C for 5 days. Brown color formation was analyzed after 1 week at 4°C. WT, loss-of-ribosomal DNA-silencing (LRS), and met15 null strains are shown as controls.

FIG. 4.

Transcriptional silencing assays for reporter strains expressing substitutions at histone H4 K59 and K31. (A) Cells were plated and analyzed as for Fig. 3. (B) ribosomal DNA silencing was monitored by plating and analysis as for Fig. 3.

FIG. 5.

Acetylation of H3 K56 in S. cerevisiae. (A) Silencing reporter strains expressing alanine, glutamine, and arginine substitutions at histone H3 K56 were plated and analyzed as for Fig. 3. (B) ribosomal DNA silencing strains were plated and analyzed as for Fig. 3. WT, loss-of-ribosomal DNA-silencing (LRS), and met15 null strains are shown as controls. (C) MS/MS spectrum of the doubly charged lysine acetylated peptide ion, FQK_acetylSTELLIR. The complete sequence including the acetylated lysine residue was deduced from the y-ion series as shown. Diagnostic fragment ions (53) for an acetylated lysine residue are observed at m/z 84.1 and m/z 126.1. (D) The MALDI postsource decay spectrum of a histone H3 peptide isolated from a trypsin-digested total yeast histone preparation. The peptide was N-terminally sulfonated prior to mass spectrometric analysis.

FIG. 6.

The DNA repair response is affected by substitutions of core nucleosome residues. (A) Strains expressing mutated histones were plated with an initial OD₆₀₀ of 0.5 and serially diluted fivefold on SC as a growth control and SC plus 200 mM HU to analyze the ability of these cells to recover from DNA damage-induced stress. A rad52 null strain was used as a positive control. (B) Cells harboring substitutions at H3 K56 were examined on plates containing CPT at the indicated concentrations. The initial density of cells corresponded to an OD₆₀₀ of 0.5, and cells were serially diluted fivefold.