The H2A substitutions affect histone levels on genes but do not greatly affect transcription of genes that are sensitive to nucleosome occupancy.

<p><b>(A)</b> Western analysis of H2B, H2A, and H3 levels in the H2A mutant strains. G6PDH levels served as a loading control. <b>(B, C, D)</b> Analysis of H2B (KY2674), H2A (KY2675), and H3 (KY943) occupancy at the 5’- and 3’ ends of <i>PYK1</i> and at <i>TELVI</i> by ChIP. The error bars denote SEM of three independent experiments. <b>(E)</b> Northern analysis assessing the effects of the H2A substitutions and H2B-K123R (KY2044) on <i>SER3</i>, <i>SRG1</i>, <i>FLO8</i> and <i>FLO8</i> cryptic transcript levels. Upper band (*) corresponds to the full-length <i>FLO8</i> transcript and the lower band (**) corresponds to the cryptic internally initiated transcript. The <i>spt6-1004</i> (KY2678) and <i>spt16-197</i> (KY2679) temperature-sensitive alleles serve as positive controls for cryptic initiation and <i>SER3</i> derepression and are isogenic to the wild-type strain KY2677. <i>SCR1</i> was used as a loading control.</p

Recruitment of histone modification and elongation machinery is impaired in the H2A mutants.

<p>ChIP analyses of HSV-Bre1 (KY2674) <b>(A)</b>, Rtf1 (KY2674) <b>(B)</b>, Spt5 (KY943) <b>(C)</b>, Spt16 (KY2675) <b>(D)</b>, and HSV-Set1 (KY2719) <b>(E)</b> at the 5’- and 3’-ends of transcribed loci (<i>PYK1</i> and <i>PMA1</i>) and at <i>TELVI</i>. The error bars represent SEM of three independent experiments.</p

Deletion of <i>UBP8</i> variably affects the recovery of H2B K123ub, H3 K4me³, and H3 K79me^2/3 in the H2A mutants.

<p><b>(A)</b> Western analysis of H2B K123ub and total H2B in the indicated wild-type and H2A mutant strains. <b>(B)</b> The relative levels of H2B K123ub between <i>ubp8∆</i> (KY2086) and <i>UBP8</i> (KY943) backgrounds are shown. The ratio of H2B K123ub in <i>ubp8∆</i> to H2B K123ub in <i>UBP8</i> was calculated after normalizing to total H2B levels. To determine the fold change of H2B K123ub levels between H2A mutants, these ratios were normalized to the wild-type H2A background. Error bars represent SEMs of three biological replicates. (C) Western analysis of H3 K4me³ and H3 K79me^2/3 levels in H2A mutants in the presence or absence of <i>UBP8</i>. Total H3 served as a loading control.</p

Summary of molecular defects tested in the H2A mutants.

<p>Phenotypes (listed on the left) of the histone mutants rated relative to wild type (WT) and additional controls, which are defined in the figure (right column). Molecular defects not determined for specific mutants are denoted by "N.D."</p

Substitutions in H2A cause H2B K123ub defects.

<p><b>(A)</b> Western analysis of H2B K123ub, as well as total H2B and G6PDH, both of which served as loading controls. KY1599 (<i>rtf1∆</i>) was used as a negative control. The bar graph shows H2B K123ub levels normalized to total H2B levels. These relative H2B K123ub levels were normalized to the wild-type value. Error bars represent SEM of three independent experiments. <b>(B)</b> ChIP analysis of H2B K123ub occupancy at the 5'- and 3'- ends of <i>PYK1</i> and <i>PMA1</i> and at a nontranscribed region, <i>TELVI</i>. H2B K123ub ChIP values were normalized to total H2B ChIP values. The error bars represent SEM of three independent experiments.</p

Transcription elongation is affected by substitutions in the nucleosome acidic patch.

<p><b>(A)</b> Diagram of experimental procedure. Cells were grown in medium containing 2% galactose (zero time point) and then 2% glucose was added to shut off transcription. Samples were taken at zero, two, four, and eight-minute time points for cross-linking. ChIP of the Rpb3 subunit of Pol II across <i>YLR454W</i> was performed in <b>(B)</b> wild type (WT), <b>(C)</b> H2A-E65A, <b>(D)</b> H2A-L66A, <b>(E)</b> H2A-E93A, and <b>(F)</b> H2B-K123R strains, which were transformants of KY2676. Values were normalized to the zero time point for each locus. Error bars represent SEM of three biological replicates.</p

The H2A substitutions differentially affect H3 methylation.

<p><b>(A)</b> Western blots were probed with antibodies to detect di- and tri-methylation of H3 K4, K36, and K79 as indicated. Total H3 and G6PDH levels were used as loading controls. Strains lacking <i>SET1</i> (KY1715), <i>DOT1</i> (KY1717), and <i>SET2</i> (KY1716) show the specificity of the antibodies used. <b>(B, C)</b> ChIP analysis of methylated H3 K79 and K4 at <i>PYK1</i>, <i>PMA1</i> and <i>TELVI</i>. The H3 K79 antibody used in these experiments can detect both the di- and tri-methylated states (Abcam). The error bars represent SEM of three independent experiments.</p

Identification of H2A and H2B residues required for growth in the absence of <i>RKR1</i>.

<p><b>(A)</b> Synthetic lethal/sick phenotypes of <i>rkr1∆ hta1</i> and <i>rkr1∆ htb1</i> mutants were assessed through ten-fold serial dilution assays. Double mutant cells, as well as control <i>RKR1 hta1</i> and <i>RKR1 htb1</i> cells, were plated on SC-His medium as a growth control and on SC-His + 5-FOA medium to select for histone mutant plasmids and against the <i>URA3</i>-marked <i>HTA1-HTB1</i> plasmid. Library plasmids were transformed into the <i>rkr1∆</i> strain KY981 and wild-type strain KY943. KY2265 and KY2249 were used as respective negative and positive growth controls on 5-FOA plates. (<b>B)</b> X-ray crystal structure of the nucleosome, denoting histones H2A, H2B, H3, and H4 in cyan, green, yellow, and white, respectively. As depicted in red, the majority of histone residues identified in the <i>rkr1∆</i> synthetic lethality screen form a surface-exposed patch on the nucleosome. <b>(C)</b> Electrostatic potential (red is negative, blue is positive) of the nucleosome core particle. This figure was created using Pymol (PDB 1ID3 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005420#pgen.1005420.ref035" target="_blank">35</a>]).</p