variation between BY and RM has been extensively studied in the same growth conditions as here [20], thus allowing direct examination of this possibility. We considered three regions at the beginning, middle and end of genes, and computed in each one the average log-ratio of H3K14 acetylation between the two strains. In all three regions this ratio was clearly not correlated to expression differences (Figure 3A). Consistently, SNEPs acetylated in the strain with highest gene expression were not over-represented in any of the three regions (Figure 3B). The two strains therefore have a high degree of divergence at both transcriptomic and epigenomic levels but with no apparent connection between the two. BYac SNEPs are more abundant in conserved regulatory regions If not correlated to expression differences, do SNEPs have any functional implication? If so, one might expect them to target nucleosomes located at critical positions for gene regulation, such as nucleosomes containing a transcription factor binding site. In favor of this, and in accordance with the distribution pattern described above, we found a striking (3.2-fold) enrichment of BYac SNEPs in nucleosomes that fully covered a conserved regulatory site [35] (Figure 3C). BYac SNEPs were also abundant in conserved non-coding regions regardless of regulatory sites (Figure 3C). In contrast, RMac SNEPs were poorly present at these conserved regions (Figure 3C), which is consistent with their enrichment within protein coding regions (Figure 2D). The abundance of BYac SNEPs at conserved regulatory sites indicates that genetic and epigenetic polymorphisms can be complementa- ry, the latter providing diversity where the former is more constrained. SNEPs are enriched in genes with high expression variability Are genes with high inter-strain variability in gene expression the same genes as those having high epigenomic variability? Although SNEP acetylation was not associated to higher gene expression, it remained possible that genes with high expression changes contained more SNEPs than others. We examined this possibility by ranking genes according to their BY/RM expression fold-change and by counting their SNEP content (Figure S6A). This showed that indeed, SNEPs were more frequent in genes showing high inter-strain transcriptional differences. Several studies have examined the evolvability of yeast gene expression levels. For example, when comparing 4 yeast species across 5 different stressful environments, Tirosh et al. showed that genes can have very different inter-species expression divergence [36]. Similarly, Landry et al. showed that S. cerevisiae genes greatly differ in their divergence of expression across independent mutation accumulation lineages [37]. To see if SNEP abundance was correlated with expression evolvability beyond the scope of the BY and RM strains, we used these datasets to rank genes either by their expression divergence [36] or by their mutational variance [37]. This showed an unambiguous association between SNEP frequency and expression variability (Figure S6B and S6C). The extent of gene x environment interactions in the control of gene expression has been thoroughly estimated by Smith et al. who used the same BY and RM strains as here and compared their transcriptomes between two different steady-state environ- ments: growth in glucose and growth in ethanol [38]. Using this dataset, we examined if SNEP frequency in genes was associated with the level of genotype x environment interaction in the gene’s expression level (Figure S7). We found that BYac but not RMac SNEPs were more frequent in genes with high genotype x Single Nucleosome Epi-Polymorphisms environment interaction, with no correlation between the direction of the SNEP (which strain is acetylated) and the direction of the interaction (which strain shows the highest change between glucose and ethanol growth). SNEP acetylation was therefore not predictive of the amplitude of expression change between the two different environments. However, it is important to note that these two environments were stable and this dataset did not correspond to the dynamic response to an environmental change. Expression variability within a given strain background has also been studied in a broad sense by estimating the extent of variation across a large compendium of environmental conditions and/or specific genetic perturbations [36]. This ‘‘transcriptional plasticity’’ varies greatly among genes. For example, housekeeping genes display very low plasticity as they present stable expression across many conditions. This plasticity was previously associated with expression evolvability [36,37] and nucleosome occupancy at promoter regions [39]. Using the values previously compiled [36], we found that SNEPs were enriched in genes displaying high transcriptional plasticity (Figure 4A). This enrichment was also visible when considering only SNEPs with strong effect (1.4-fold acetylation difference). Genes with at least one H3K14ac SNEP had significantly higher plasticity than genes with no SNEP (t-test P = 3.661027 and 1.261026 for BYac and RMac SNEP, respectively). Finally, to see if SNEPs were more frequent among nucleosomes known to be evicted upon an environmental change, we used pre- vious maps of nucleosome positioning in normal and stress con- ditions [26] and counted SNEPs among 147 remodeled nucleosomes and 61,623 unperturbed ones. Although this dataset represents only one environmental change and the remodeling of relatively few nucleosomes, a significant 1.7 fold enrichment of SNEPs was seen among these ‘mobile’ nucleosomes (P = 0.01, Chi-square). One SNEP is associated with differential gene activation upon stimulation We reasoned that SNEPs could influence the dynamics of activation or repression. Intuitively, an acetylated nucleosome may be more rapidly evicted than a non-acetylated one upon promoter activation [40]. We noticed one SNEP where association with a differential dynamic response could be tested experimentally. A nucleosome contained a binding site for transcription factor Hsf1 (Heat Shock Factor 1) in the promoter region of the AHA1 gene, which codes for a co-chaperone of Hsp90 known to be activated upon heat- shock [41]. This nucleosome had similar positioning in BY and RM but was acetylated at H3K14 in BY only (Figure 4B), while the DNA sequence of Hsf1-binding site was fully conserved between the two strains. Notably, other nucleosomes of the region were acetylated in both strains. We exposed BY and RM cells to heat shock and monitored AHA1 mRNA by real-time quantitative RT-PCR (Figure 4C). Gene induction was unambiguously more pronounced in BY than RM. This marked difference was not observed when quantifying mRNA from three other HSP genes (SSA3, FES1 and CPR6) lacking SNEP (Figure S8). This example illustrates how one SNEP can be associated with gene activation differences upon an environmental change. Discussion We observed that nucleosome positioning at promoter regions was similar between two unrelated strains of S. cerevisiae. Because these strains have a large extent of transcriptional differences, this argues that differences in nucleosome occupancy profiles are not a major source of intra-species variation in gene expression. PLoS Genetics | www.plosgenetics.org 5 April 2010 | Volume 6 | Issue 4 | e1000913