Single Nucleosome Epi-Polymorphisms density distribution of genes according to this measure, from actual data (colored) and data where indexes of expression ratios were permuted (black). Colored curves are not significantly shifted to the right (as compared to black curves), ruling out association between SNEP and gene expression differences. (C) BYac but not RMac SNEPs are more abundant at conserved regulatory sites. Nucleosomes were divided in three categories: nucleosomes that covered entirely a conserved regulatory site from the list of MacIsaac et al. [35], nucleosomes that did not contain such sites but were located in highly conserved non-coding sequences (see Methods), and nucleosomes excluded from the first two categories. The fraction of SNEPs within each category is presented. Error bars, 95% C.I. The 3.2 and 2.6 fold enrichment at regulatory sites and other conserved regions, respectively, were highly significant (P,2.2610216). doi:10.1371/journal.pgen.1000913.g003 In contrast, the epigenomic profile of H3K14ac was highly variable and this variability targeted specific nucleosomes. The presence/absence of a modification at a particular nucleosome in a given cell is, by definition, a discrete state. However, we observed quantitative acetylation differences that were often subtle (1.2 to 1.5 fold). This is likely due to high cell-to-cell heterogeneity and high dynamics of the acetylated state: all states from billions of cells were averaged in our samples, and no dynamical information was acquired over time. It is therefore important to interpret SNEPs as differences in the overall acetylation level across a cell population and not as a uniform epigenotype shared by all cells of the sample. Natural epigenetic variation was previously reported at the level of methylated DNA (meDNA), particularly in plants [3,14]. In this case also, differences were not necessarily discrete but often continuous. Important properties of SNEPs distinguish them from meDNA epi-polymorphisms. Methylated epi-alleles were predic- tive of lower gene expression [14] but SNEPs with reduced acetylation were not. In addition, no evidence was reported on a possible role of meDNA variation on the dynamics of gene activation. Since histone-tail modifications are known to be highly reversible and dynamic, the basis and the origin of SNEPs remain to be further investigated. We observed that the two strains had different overall patterns of acetylation along genes, with a preferential acetylation near TSS and TES in the BY strain, while the RM strain had enriched acetylation in the second half of transcribed regions. This pattern difference accounted for many SNEPs and may result from trans-acting factors that act differentially in the two strains. However, 1806 SNEPs could not be attributed to this general inter-strain difference. Focusing on these SNEPs only, we looked again at their genomic distribution, their potential correlation to expression divergence and enrich- ment in genes with high plasticity (Figure S9). All conclusions made in our study were retrieved for this subset of SNEPs. Thus, the differential pattern of acetylation does not explain the general SNEP properties. Nucleosomal epi-polymorphisms may offer an Figure 4. SNEP correlation with transcriptional plasticity. (A) SNEP density is correlated to transcriptional plasticity genome-wide. 4,232 genes were ranked according to their plasticity values from Tirosh et al. [36] on the x-axis. BYac and RMac SNEP frequencies were counted among nucleosomes located in each gene (coding region plus and minus 250 bp), and averaged in 500-genes sliding windows (y-axis). (B) Scheme of nucleosome organization in the regulatory region of the AHA1 gene. Rectangles represent nucleosomes, colored according to the mean log(ac/nuc) value across all probes of the nucleosome. SNEP detection (-log10(P-value)) is indicated above each nucleosome, with significance cutoff indicated as a dashed line. A highly significant SNEP covers the DNA binding site for HSF1 transcription factor (blue spot). Arrow, transcription start site. Brown box, beginning of coding sequence. (C) Differences in kinetics of AHA1 mRNA expression during heat-shock. Four independent experiments were performed. In each case, cultures of exponentially growing BY (red) and RM (black) cells were shifted from 30uC to 37uC, and cells were collected at indicated times after the transition. AHA1 mRNA was quantified by quantitative real-time PCR with reverse-transcription relative to ACT1 mRNA (a gene known to be stable after heat shock). Bars: +/2 standard deviations. doi:10.1371/journal.pgen.1000913.g004 PLoS Genetics | www.plosgenetics.org 7 April 2010 | Volume 6 | Issue 4 | e1000913