Divergence and selectivity of expression-coupled histone modifications in budding yeasts.

Various histone modifications are widely associated with gene expression, but their functional selectivity at individual genes remains to be characterized. Here, we identify widespread differences between genome-wide patterns of two prominent marks, H3K9ac and H3K4me3, in budding yeasts. As well as characteristic gene profiles, relative modification levels vary significantly amongst genes, irrespective of expression. Interestingly, we show that these differences couple to contrasting features: higher methylation to essential, periodically expressed, 'DPN' (Depleted Proximal Nucleosome) genes, and higher acetylation to non-essential, responsive, 'OPN' (Occupied Proximal Nucleosome) genes. Thus, H3K4me3 may generally associate with expression stability, and H3K9ac, with variability. To evaluate this notion, we examine their association with expression divergence between the closely related species, S. cerevisiae and S. paradoxus. Although individually well conserved at orthologous genes, changes between modifications are mostly uncorrelated, indicating largely non-overlapping regulatory mechanisms. Notably, we find that inter-species differences in methylation, but not acetylation, are well correlated with expression changes, thereby proposing H3K4me3 as a candidate regulator of expression divergence. Taken together, our results suggest distinct evolutionary roles for expression-linked modifications, wherein H3K4me3 may contribute to stabilize average expression, whilst H3K9ac associates with more indirect aspects such as responsiveness

Interspecies differences in H3K4me3, compared to H3K9ac, better predict expression divergence.

<p><b>(A)</b> Correlation between interspecies expression differences and either H3K9ac (<i>red</i>) or H3K4me3 (<i>blue</i>) changes, at different regions along a gene, as indicated. Correlations were calculated for significant changes in expression and modifications (absolute differences greater than 0.4 (log₂ scale)). <b>(B)</b> Scatter plot of interspecies differences in mRNA levels against changes in ‘peak’ H3K9ac. Genes showing consistent changes between <i>S. cerevisiae</i> and <i>S. paradoxus</i> (<i>dark red</i>), opposite changes (<i>pink</i>), no changes in either parameter (<i>light grey</i>), changes in only in H3K9ac (<i>light red</i>), and changes only in expression (<i>dark grey</i>) are marked. The numbers of genes showing consistent or opposite changes are noted, and the relative proportions of each group are indicated in the adjacent pie chart. <b>(C)</b> As in (B), but for H3K4me3 changes. Genes showing consistent changes (<i>dark blue</i>), opposite changes (<i>light blue</i>), no changes in either parameter (<i>light grey</i>), changes in only in H3K9ac (<i>blue</i>), and changes only in expression (<i>dark grey</i>) are marked. <b>(D)</b><i>Upper panel</i>, Interspecies correlation between expression differences and H3K9ac (<i>red</i>) or H3K4me3 (<i>blue</i>) differences at different regions along a gene, as indicated, calculated at increasing absolute thresholds (for both expression and modification changes). <i>Middle panel</i>, Graph depicting the proportion of genes for which interspecies changes in expression and modifications (at different regions) are consistent (in the same direction). Percentages were calculated for an increasing threshold of absolute modification differences, and considering significant expression differences (> 0.4). <i>Lower panel,</i> Genes with the largest and smallest differences in expression <i>S. cerevisiae</i> and <i>S. paradoxus</i> (‘<i>divergent</i>’ and ‘<i>non-divergent</i>’; 1000 genes each) were taken. Thereafter, for an increasing threshold of absolute differences in modifications at different regions, the fold enrichment of divergent over non-divergent genes was calculated at each threshold. <b>(E)</b> Enrichment (<i>upper panels</i>) or depletion (<i>lower panels</i>) of sets of genes defined according to patterns of H3K9ac/mRNA changes (<i>left</i>) or H3K4me3/mRNA changes (<i>right</i>) amongst the indicated architectural or phenomenological gene classes; <i>p</i> values were calculated using a hypergeometric test. <b>(F)</b> Genes were classified based on interspecies variation in all three parameters (H3K9ac, H3K4me3, expression): those showing no change in either (<i>light grey</i>), consistent changes amongst all three (<i>purple</i>), consistent changes between H3K9ac and H3K4me3 but not expression (<i>dark grey</i>), consistent changes between H3K9ac and expression but not H3K4me3 (<i>light red</i>), and those showing consistent changes between H3K4me3 and expression but not H3K9ac (<i>blue</i>). Gene sets were subsequently analyzed as in (E).</p

Divergence of H3K9ac or H3K4me3 patterns between closely related yeasts.

<p><b>(A)</b> Overall correlation of H3K9ac and H3K4me3 patterns between <i>S. cerevisiae</i> and <i>S. paradoxus</i>. Heatmap of Pearson correlations calculated after juxtaposing modification profiles (mean signal over 20bp intervals) at c.6000 orthologous genes (including the promoter and coding regions). <b>(B)</b> Average H3K9ac (<i>upper left panel</i>) and H3K4me3 (<i>upper right panel</i>) profiles around the TSS and TTS, for <i>S. cerevisiae</i> (<i>dark green</i>) and <i>S. paradoxus</i> (<i>light green</i>). Interspecies correlations for H3K9ac and H3K4me3 at the given positions along a gene are shown in the lower panels. <b>(C)</b> Interspecies differences (log₂(Cer/Par)) in modification levels for different regions along a gene (<i>upper panel</i>). Mean levels across three regions were taken: the promoter (‘<i>prom</i>’: −320 to −160 relative to the TSS), the respective loci with highest prevalence on average (‘<i>peak</i>’: 0 to +140 for H3K9ac, +100 to +580 for H3K4me3) and around the TTS (‘<i>end</i>’: −260 to +60 relative to the TTS), as indicated. The percentage of genes with absolute differences at these regions exceeding an increasing threshold (log₂ scale) is depicted. <b>(D)</b> Comparison of intragenic differences in modification levels (log₂(H3K9ac/H3K4me3)) between <i>S. cerevisiae</i> and <i>S. paradoxus</i> at orthologous genes. The interspecies correlation, and a linear fit of the data are shown. <b>(E)</b> Relative modification levels at orthologous genes for selected ontological groups. Shown are interspecies comparisons for ‘protein synthesis’ and ‘cytoskeleton’ genes (<i>upper panel</i>), and ‘ribosome’ and ‘RiBi’ genes (<i>lower panel</i>). <b>(F)</b> Scatter plots comparing ‘peak’ H3K9ac and ‘peak’ H3K4me3 between species. For each modification, the most divergent genes were extracted by applying the Lowess method to the interspecies plot, and selecting those genes farthest from the regression curve in either direction (c. 800 genes each; <i>upper left</i> and <i>lower right</i> panels). Genes with higher H3K9ac in <i>S. cerevisiae</i> (<i>dark green</i>) or <i>S. paradoxus (light green)</i> were overlaid onto the interspecies H3K4me3 plot (<i>upper right</i>), or vice versa (<i>lower left</i>). <b>(G)</b> Plot of interspecies differences in H3K9ac against H3K4me3. Genes showing consistent changes for both modifications (<i>black</i>), opposite changes (<i>grey</i>), no changes in either modification (<i>light grey</i>), changes in only in H3K9ac (<i>pink</i>), and changes only in H3K4me3 (<i>light blue</i>) are marked. Absolute differences above 0.4 (log₂ scale) were considered as significant. The Pearson correlation and a linear fit for the data are shown. Relative proportions of each group are indicated in the adjacent pie chart.</p

Biased distributions of expression-associated histone modifications in <i>S. cerevisiae</i>.

<p><b>(A)</b> Average H3K9ac (<i>red</i>) and H3K4me3 (<i>blue</i>) profiles around the transcription start and termination sites of genes (<i>TSS</i> and <i>TTS</i>, respectively). <b>(B)</b> Traces depicting the correlation of H3K9ac and H3K4me3 profiles with nucleosome occupancy, around the TSS and TTS of genes. <b>(C)</b> Bar graphs showing the correlation between mRNA expression and modifications at individual nucleosomes across genes, as indicated. <b>(D)</b> Levels of H3K9ac (at +1nuc) and H3K4me3 (mean of +2nuc and +3nuc levels) were obtained for all genes. Genes were then grouped by gene ontology (‘GO slim’ categories, <a href="http://www.geneontology.org" target="_blank">www.geneontology.org</a>) or by pre-defined transcriptional modules <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101538#pone.0101538-Ihmels1" target="_blank">[30]</a>, and the mean H3K9ac and H3K4me3 per category plotted against each other. Gene groups exhibiting higher average levels of one modification over the other are indicated. <b>(E)</b> Modification levels at specific nucleosomes for all genes were taken as in (D), and the ratio between H3K9ac and H3K4me3 calculated (log₂(H3K9ac/H3K9ac)). Genes were ranked according to this ratio, and three sectors (1200 genes each) were considered: H3K4me3 > H3K9ac (<i>blue</i>), H3K4me3 ≈ H3K9ac (<i>grey</i>), and H3K4me3 < H3K9ac (<i>red</i>). Enrichment of various categories of genes (as in (D)) within each of these sectors was then assessed using a hypergeometric test. Significantly enriched categories (−1*log₁₀(pval) > 2) are depicted in the bar graphs. <b>(F)</b> Genes were classified according to their promoter nucleosome architecture (occupied proximal nucleosome, <i>‘OPN’</i>; depleted proximal nucleosome, <i>‘DPN’</i>), or according to whether or not they incorporate a TATA-box within the promoter (TATA-containing, <i>‘TATA’</i>; or TATA-deficient, <i>‘Tless’</i>). Thereafter, enrichment (<i>top panel</i>) or depletion (<i>bottom panel</i>) of these classes amongst the sectors defined in terms of the genic H3K9ac/H3K4me3 ratio (as in (E)) was calculated using a hypergeometric test (<i>top panel)</i>. Calculated p values are shown as -1*log10(pval)). The number of genes in each subgroup is indicated. <b>(G)</b> As in (F) but assessing the enrichment (<i>top panel</i>) and depletion (<i>bottom panel</i>) of genes classified according to several features of expression: ‘<i>responsive</i>’ and ‘<i>non-responsive</i>’ genes, defined by their expression variance across a large compendium of conditions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101538#pone.0101538-Ihmels1" target="_blank">[30]</a>; ‘<i>periodic</i>’ genes, which show cyclical expression between successive cell cycles (800 genes; as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101538#pone.0101538-Pramila1" target="_blank">[29]</a>); ‘<i>essential</i>’ and ‘<i>non-essential</i>’ genes (defined according to the viability in rich media of their respective deletion mutants).</p

Hsp90 restrains ErbB-2/HER2 signalling by limiting heterodimer formation

ErbB-2/HER2 is an oncogenic tyrosine kinase that regulates a signalling network by forming ligand-induced heterodimers with several growth factor receptors of the ErbB family. Hsp90 and co-chaperones regulate degradation of ErbB-2 but not other ErbB members. Here, we report that the role of Hsp90 in modulating the ErbB network extends beyond regulation of protein stability. The capacity of ErbB-2 to recruit ligand-bound receptors into active heterodimers is limited by Hsp90, which is dissociated from ErbB-2 following ligand-induced heterodimerization. We show that Hsp90 binds a specific loop within the kinase domain of ErbB-2, thereby restraining heterodimer formation and catalytic function. These results define a role for Hsp90 as a molecular switch regulating the ErbB signalling network by limiting formation of ErbB-2-centred receptor complexes