Dissecting Nucleosome Function with a Comprehensive Histone H2A and H2B Mutant Library

Abstract

Using a comprehensive library of histone H2A and H2B mutants, we assessed the biological function of each amino acid residue involved in various stress conditions including exposure to different DNA damage-inducing reagents, different growth temperatures, and other chemicals. H2B N- and H2A C-termini were critical for maintaining nucleosome function and mutations in these regions led to pleiotropic phenotypes. Additionally, two screens were performed using this library, monitoring heterochromatin gene silencing and genome stability, to identify residues that could compromise normal function when mutated. Many distinctive regions within the nucleosome were revealed. Furthermore, we used the barcode sequencing (bar-seq) method to profile the mutant composition of many libraries in one high-throughput sequencing experiment, greatly reducing the labor and increasing the capacity. This study not only demonstrates the applications of the versatile histone library, but also reveals many previously unknown functions of histone H2A and H2B.

Keywords: DNA damage; heterochromatin gene silencing; histone; post-translational modification.

Figure 1

High-throughput phenotype analysis of a yeast library containing two copies of histone mutants. (A) More HU-sensitive mutants were identified with the BY-H2ML1&2 library. The reported mutants (Matsubara et al. 2007; Sakamoto et al. 2009) were marked in bright red for H2A and bright green for H2B. For BY-H2ML1&2, H2A/H2B mutants were marked with different red/green colors, indicating the strength of phenotype, which was defined by using a number from 1 to 4 (to indicate weak to strong phenotype). Bright red: H2A mutants with phenotypes classified 3 or 4. Russet-red: H2A mutants with phenotypes classified 1 or 2. Bright green: H2B mutants with phenotypes classified 3 or 4. Dull green: H2B mutants with phenotypes classified 1 or 2. If two or more mutants for a certain residue showed phenotypes, the highest phenotype degree of this residue was displayed. H2A R30 is not shown since it cannot be visualized at this angle. The S. cerevisiae nucleosome structure is used in the entire paper (PDB number: 1ID3). The locations of corresponding clusters of mutants in the nucleosome structure were marked with yellow lines and circles. (B) Venn diagram of mutants sensitive to different DNA damage-inducing stresses. (C) Statistical analysis of mutants with phenotypes according to their biological processes. See the classification of different stresses in Table S2 in File S1. I: mutants with one class of phenotype, blue; II: mutants with two classes of phenotypes, light green; III: mutants with three classes of phenotypes, yellow; and IV: mutants with four classes of phenotypes, orange. (D) Evolutionary conservation scores for mutants with different classes of phenotypes were calculated using ConSurf. The definition of phenotype numbers I–IV is the same as those in (C). Data were represented as mean ± SEM. * P < 0.05 and ** P < 0.01. CPT, camptothecin; MMS, methyl methanesulfonate; HU, hydroxyurea; UV, ultraviolet.

Figure 2

Phenotype analysis for histone tails of H2A and H2B. (A) The phenotypes of deletion mutants of H2B N terminal tail; (B) The phenotypes of deletion mutants of H2A C terminal tail; (C) The phenotypes of deletion mutants of H2A N terminal tail; (D) The phenotypes of deletion mutants of H2B C terminal tail. For simple visualization, the amino acids within histone tails were divided into different units based on the mutagenesis strategy. The numbers of phenotypes for these mutants are marked with the same colors used in Figure 1C. I: mutants with one class of phenotype, blue; II: mutants with two classes of phenotypes, light green; III: mutants with three classes of phenotypes, yellow; and IV: mutants with four classes of phenotypes, orange. The lethal mutant H2A del118-127 is marked with a brown color and red characters. Mutants without any phenotypes are shown in gray. Point mutants with phenotypes within these histone tails are also labeled.

Figure 3

Identification of histone H2A and H2B mutants affecting genome stability. (A) The frequency of chromosome (Chr) loss for the indicated histone H2A mutants (black) and H2B mutants (orange). (B) The distribution of mutants with elevated frequency of chromosome loss in four core histones. Mutants in histone tails and the compound substitutions are not shown. The picture in the upper right side is the top view of the nucleosome and the lower one is the lateral view. H2A/H2B mutants are marked in bright red and H3/H4 mutants are marked in bright green.

Figure 4

Identification of histone mutants affecting heterochromatin silencing. (A) Histone H2A and H2B mutants with LTS, except tail deletion mutants and the compound substitutions, are highlighted in the nucleosome structure. Bright red: H2A mutants. Bright green: H2B mutants. (B) Histone H2A and H2B mutants with LRS, except tail deletion mutants and the compound substitutions, are highlighted in the nucleosome structure with the same colors as in (A). (C) Telomere silencing defects of the histone H2A C-terminal tail deletion mutants. Dark gray lines represent the H2A C-terminal tail. The number of corresponding amino acids are marked above. The deleted regions are marked with orange dotted lines. (D) Telomere silencing phenotypes of the histone H2B N-terminal tail deletion mutants. Dark gray lines are used to represent H2B N-terminal tail. The number of corresponding amino acids are marked above. The deleted regions are marked with orange dotted lines as (C). ITS, increased telomere silencing; LRS, loss of rDNA silencing; LTS, loss of telomere silencing; WT, wild-type.

Figure 5

MMS sensitivity assay of histone mutants with barcode sequencing. (A) A schematic diagram to analyze stress sensitivities of pooled histone mutants by bar-seq. The mutants used in this assay were strains with a single copy of integrated histone genes (BY-H2ML1). (B) Correlation of the two replicates each for the Exp and Ctrl groups is indicated. (C) The sequence results for one of the MMS-treated mutant populations. The mutants with a log2 ratio below −1 were arbitrarily defined as MMS-sensitive candidates. (D) Comparison of identified MMS mutants identified by bar-seq and SD test for individual strains. The mutants used in both tests were strains with a single copy of integrated histone genes (BY-H2ML1). (E) Confirmation of MMS sensitivity of candidates identified by high-throughput sequencing using the SD test. bar-seq, barcode sequencing; Ctrl, control; Exp, experimental; MMS, methyl methanesulfonate; SD, serial dilution; WT, wild-type; YPD, yeast extract peptone dextrose.