Histone H3-K42A alters the S. cerevisiae transcriptome. (A) Kinetics of appearance of MET16 mRNA in cells expressing wild-type (WT) or K42A histone H3, as described in the Materials and Methods. Error bars represent the standard deviation between two biological replicates of each sample, each assayed in triplicate. (B) Table summarizing RNA length changes detected in H3-K42A cells using yeast genomic tiling array as described in the Materials and Methods. Four classes of mRNA length changes were analyzed, and the number of transcripts in K42A cells that fall into each class is indicated. (C) Venn diagram representing the number of genes within each class of RNA length change. The overlap regions indicate the counts of loci showing aberrant RNA length changes that can be classified by two distinct transcriptional events. (D) Integrated Genome Browser screen shot of log₂ wild-type (WT) and H3-K42A RNA levels, as well as the differential RNA levels (log₂ K42A/WT) for a segment of chromosome 2 spanning YBR040W. The horizontal bars marked with asterisks represent aberrant length changes, indicating both 5′ extension (**) and 3′ extension (*) events at this locus.

Histone H3-K42 is dimethylated in S. cerevisiae. (A) Electrospray tandem mass spectrum (MS/MS) of the doubly charged dimethylated peptide: YK(dimet.)PGTVALR, m/z 516.869, recorded using a quadrupole time-of-flight mass spectrometer in an LC-MS/MS experiment (Agilent 1200 series CapLC coupled to QSTAR Pulsar). It was possible to confirm the site of modification using the doubly charged y ions y7 and y8. The spectrum was further confirmed by comparison with the corresponding unmodified peptide, which was also subjected to MS/MS (data not shown). (B) Dot blot analysis of anti-H3-K42me2 serum. The unmodified and K42me2-modified H3 peptides were spotted onto the PVDF membrane and probed with anti-H3-K42me2 serum in the presence of increasing amounts of either the competing modified or unmodified peptide. (C) Western blot analysis. Recombinant Escherichia coli expressed histone H3 or whole-cell extracts (WCE) prepared from yeast expressing wild-type (WT) or various substituted histone H3 were run on a gel and probed with either anti-H3-K42me2 serum or anti-histone H3 C terminus antibody. Peptide competition of the H3-K42me2 band was done in the presence of K42 mono-, di-, or trimethylated synthetic peptides. (D) Genome-wide histone H3-K42me2 ChIP in wild-type yeast was undertaken as described in the Materials and Methods and analyzed on yeast tiling array. The abundance of this modification across a “standard” yeast gene is indicated relative to the transcription start site (TSS) and transcription end site (TES).

Histone H3-K42A is a pleiotropic mutation, which disrupts a structurally important nucleosome surface. (A) The crystal structure of the S. cerevisiae nucleosome highlighting histone H3 Lys 42 (orange) positioned at the DNA entry and exit points. The figure was generated using PYMOL. (B) Nucleosome crystal structure surrounding H3-K42 (yellow), indicating hydrogen bonds between the lysine side chain and DNA. (C) Predicted structures of H3-K42A mutation in its nucleosomal context. (Red) Residue 42; (blue) DNA; (green/yellow) histone H3. Shown is the single rotamer that exists for this mutation. Graphics were generated using Swiss PDB viewer. (D) Growth assays were undertaken for the indicated strains as described in Materials and Methods to detect defects in transcription elongation, cell cycle, and DNA repair, and to monitor temperature sensitivity at 39°C. (E) An MVA plot indicating gene expression changes versus spot intensity of normalized microarray data of K42A versus wild-type (WT) histone H3-expressing cells.

Histone H3-K42 is an important residue during transcription elongation. (A) Strains with the indicated genotypes were plated as described in the Materials and Methods to detect transcriptional elongation defects. (B) Quantitative RT–PCR analysis of the YBR040W, YKL221W, and YNR062C transcripts using RNA prepared from cells of the indicated genotype. Expression levels in the deletion strains expressing wild-type (WT) histones were set to 1 and were compared with those from the same deletion strain harboring H3-K42A. Data are presented as fold change over wild type and represent three individual colonies of each sample analyzed in triplicate and internally normalized to ACT1 transcript levels. (*) The expression of YNR062C was not determined in the paf1Δ strain. (C) Western blot analysis of yeast whole-cell extracts from strains of the indicated genotype detecting Ser 2 phosphorylation of RNAPII CTD (top panel) and Ser 5 phosphorylation of RNAP II CTD (bottom panel). Rpb3p, a subunit of the RNAPII complex, was detected and served as a loading control. (D) ChIP analysis of H3 occupancy at both the promoter region (5′) and coding region (3′) of the loci indicated in wild-type and K42A-expressing cells. Data represent two individual colonies of each sample, each of which underwent two parallel immunoprecipitations. Quantitative PCR was used to determine the amount of DNA in each immunoprecipitation. Data were normalized internally to the levels of histone H3 at the ACT1 locus, expression of which is not affected by histone H3-K42A. Significant differences between wild type and K42A are indicated with the corresponding probability value.

Histone H3-K42me2 may play a role in transcription. (A) Strains expressing the indicated histone H3 allele were assayed for their sensitivity to 6AU to detect transcription elongation defects. ppr1Δ acts as a positive control for comparison. (B) A diagram of microarray data showing the overlap of results obtained from the hybridization of total RNA from cells expressing either K42A, K42Q, or K42L histone H3 alleles to the yeast Affymetrix chip. The expression of the genes in common between the microarray experiments are visualized with heat maps based on the clustering using Genespring software. (C) Schematic of the intragenic transcription reporter whereby HIS3 was placed downstream from the cryptic promoter in the FLO8 ORF. Plating assay to detect the ability of strains expressing the indicated histone H3 allele to initiate transcription at the FLO8 cryptic promoter. (D) Western blot analysis detecting H3-K42me2 in whole-cell extracts from strains with the indicated deletions of PAF1 complex components. Tubulin detection served as a loading control. The graph displays the quantification of H3-K42me2 band intensity as compared with tubulin levels; error bars represent the standard deviation between two biological replicates. (*) Statistically significant values.