Genomic characterization reveals a simple histone H4 acetylation code

Abstract

The histone code hypothesis holds that covalent posttranslational modifications of histone tails are interpreted by the cell to yield a rich combinatorial transcriptional output. This hypothesis has been the subject of active debate in the literature. Here, we investigated the combinatorial complexity of the acetylation code at the four lysine residues of the histone H4 tail in budding yeast. We constructed yeast strains carrying all 15 possible combinations of mutations among lysines 5, 8, 12, and 16 to arginine in the histone H4 tail, mimicking positively charged, unacetylated lysine states, and characterized the resulting genome-wide changes in gene expression by using DNA microarrays. Only the lysine 16 mutation had specific transcriptional consequences independent of the mutational state of the other lysines (affecting approximately 100 genes). In contrast, for lysines 5, 8, and 12, expression changes were due to nonspecific, cumulative effects seen as increased transcription correlating with an increase in the total number of mutations (affecting approximately 1,200 genes). Thus, acetylation of histone H4 is interpreted by two mechanisms: a specific mechanism for lysine 16 and a nonspecific, cumulative mechanism for lysines 5, 8, and 12.

Fig. 2.

K16R-specific gene expression. (A) Incremental gene expression values were plotted against each other for the residues noted. (B) K16R discriminators. ORFs having expression regulated by a specific residue mutation, independent of the mutation states of the other three residues, are shown for K16R, selected with α = 0.01 after Bonferroni correction (see Materials and Methods and Fig. 4).

Fig. 1.

Global view of gene expression in H4 tail mutants. Gene expression data for all replicates used in this study were filtered for genes changing >2-fold in at least five experiments (to avoid showing genes resulting solely from the K5,8,12,16R aneuploidies), and both experiments and genes were hierarchically clustered. (Upper) The height of the blue bar represents the number of K → R mutations in the columns below; dark blue represents mutants with K16R, and light blue represents mutants with K16 WT. (Lower) A dendrogram of the experiment cluster is shown. White boxes represent a WT K at the residue in question; black boxes represent K → R mutations. For example, the far right mutant is K8R. Arrow shows a cluster of genes regulated uniquely by K16R.

Fig. 3.

Charge counters. (A) The number of ORFs showing monotone, nonspecific response to mutations in K5, K8, and K12 was 1,267. ORFs that monotonically increase in mean expression level as a function of the number of lysine mutations (in K5, K8, and K12) are shown for confidence parameter α = 0.05 (see Materials and Methods). In total, we found 682 (+) counters and 585 (–) counters. ORFs are sorted by the geometric mean of the mean expression differences. x axis is in increasing order of number of mutations, from 1 to 3. (B) Location of chromosomal regions enriched for charge-regulated domains. We chose the 1,267 counters (using α = 0.05) and aligned them to chromosomal coordinates. Mean log ratio expression values for the average single mutant, average double mutant, and triple K5,8,12R mutant counters are shown. x axis is proportional to base-pair distance, and ORF widths are drawn to scale. The complete genomic view is shown in Fig. 9. (C) K16R has counter effects, in addition to discriminator effects, on gene expression. Gene expression data from the K5,8,12R mutant is plotted on the x axis against gene expression data from the K5,12,16R mutant on the y axis. Pink points indicate genes identified as K16R discriminators; blue points indicate the remainder of genes.