Author Summary Nucleosomes are the basic units of chromatin, with part of the long DNA molecule wrapped around a multiprotein core, which makes unpacked chromatin often portrayed as a string of pearls. This string can carry three types of sequences: DNA, methyl groups on cytosines, and, on every pearl, the presence-or-absence of histone post-translational modifica- tions such as acetylation of lysines (nucleosomal epigenome). These latter sequences can change dynamically, and the mechanisms involved are heavily studied as they participate in many physiological processes (pluripotency, disease…). However, nothing is known about the natural diversity of nucleosomal epigenomes in natural populations. As a model, we compared two unrelated yeast strains for their epigen- ome of one histone modification. We found a high divergence, which was enriched at regulatory sites and often carried on specific nucleosomes. Although this nucleosome modification is usually associated with high transcription, higher acetylation in one strain did not necessarily imply higher expression of the corresponding gene. However, one nucleosomal variation was associated with a stronger gene activation upon stimulation. These results suggest that nucleosomal epigenomes largely differ between individuals, raising essential questions on the origin of these differences and their contribution to personal responses to environmen- tal changes (such as clinical treatments). phenotypes such as cellular morphology, sensitivity to drugs, gene expression or telomere length [18–21]. BY is a commonly used laboratory strain, it is isogenic to S288c which derives from a clone isolated from a rotten fig in California. RM (also called RM11-1a) derives from an isolate collected in a Californian vineyard by Robert Mortimer [22]. We compared them with respect to nucleosome positions as well as epigenomic sequence of one histone tail modification. Nucleosome positions were mapped using whole-genome 4-bp resolution tiling microarrays [23,24]. Of the 6,553,600 probes of the microarray, 2,801,885 and 2,570,638 had a single perfect match on BY and RM genome, respectively. Only signals from these probes were used for analysis, averaging ,34 reliable probes per nucleosome. We aligned the two assembled genome sequences and used probe positions to fit a Hidden Markov Model (HMM) for inference of nucleosome positioning in each strain, as previously described [25] (Figure 1A). Note that the HMM algorithm was run on BY and RM datasets independently. Positioning looked similar between the two strains and was in very good agreement with a previously published atlas of positions [24]. We systematically aligned nucleosomes between the two strains (see Methods) and found that positioning was generally well conserved: The distance between BY and RM midpoints was smaller than 19 nucleotides in 75% of all nucleosomes; and the overlap covered at least 78% of BY nucleosome length in 80% of alignments (Figure S1). Nucleosomal occupancy was also conserved except at specific regions near heterochromatin sites (telomeres and rDNA repeat) (Figure S2). Comparison of SNP densities in linkers versus nucleosomal DNA was consistent with the results obtained when using the atlas of Lee et al. [24] (Text S1). We examined in more details occupancy around transcription start sites and found the stereotyped nucleosome-depleted regions flanked by well-positioned nucleosomes (Figure 1B and Figure S3). The typical nucleosome depletion at transcription end sites [26] was also observed in both strains (Figure S3). We clustered promoters according to their nucleosome signature in the BY strain only, and used the resulting gene order to plot occupancy Single Nucleosome Epi-Polymorphisms data in BY and RM as heatmaps, as well as differential gene expression known from previous studies (Figure 1B). The similarity of the occupancy profiles of the two strains contrasted with the large extent of transcriptional differences (Figure 1B and 1C). Inter-strain comparison of an epigenomic sequence reveals abundant nucleosome-level epi-polymorphisms We then searched for nucleosomes bearing differential levels of a specific histone post-translational modification. By analogy to nucleotide polymorphisms, we called these nucleosomes ‘Single Nucleosome Epi-Polymorphisms’ (SNEP). We chose acetylation of lysine 14 of histone H3 because it was reported to be largely distributed over the genome and not restricted to specific regulatory positions [9,27]. Hereafter, ‘BYac’ and ‘RMac’ SNEP will refer to nucleosomes where H3K14 is preferentially acetylated in BY and RM, respectively. To detect such nucleosomes, we used ChIP-CHIP [7] and we developed a custom algorithm for data analysis. First, only probes that had a single perfect match on both BY and RM genomes were retained. This precaution is important as DNA polymorphisms can greatly affect hybridization intensities. For each pair of aligned nucleosomes, probes that were not entirely covered by both BY and RM nucleosomes were also removed and a dedicated analysis of variance was applied (see Methods). The underlying linear model integrated both nucleosome mapping and chromatin immuno- precipitation experiments, which enabled to decouple the call for SNEPs from strain differences in occupancy intensity. This method identified 5,442 H3K14ac SNEPs at nominal P- value ,9.2761026 which corresponded to a False Discovery Rate [28] (FDR) of 0.0001. This list was used in all further analysis described here. SNEPs were distributed all over the genome, with few particular hotspots (Figure 2A). Epigenetic variability was very high, as these highly significant SNEPs were found in nearly 10% of nucleosomes interrogated. At the commonly used level of FDR = 0.01, 25.3% of nucleosomes were significant SNEPs, and further relaxing the detection threshold to FDR = 0.2 listed 31,854 SNEPs. We can therefore assume that about 40% of the chromatin is variable for this epigenetic mark between the two strains. In most cases, SNEPs were not detected as all-or-none nucleosomal acetylation, but as a quantitative difference between the two strains. The degree of inter-strain difference varied between SNEPs (Figure S4), with most cases displaying a 1.2 to 1.5 fold difference. Intriguingly, the acetylation difference was more pronounced in BYac SNEP (918 SNEPs at .1.5 fold) than in RMac SNEPs (274 SNEPs at .1.5 fold). Genomic distribution of SNEPs Because some highly polymorphic DNA features are associated with chromatin silencing, specific cases of histone acetylation epi- polymorphisms could be expected. One example is the rDNA locus, a repetitive sequence silenced by the Sir2 histone deacetylase [29], which is 15.6 Kb longer in RM than in BY. This higher repeat length could better recruit deacetylase activity and generate BYac SNEP in the vicinity of the repeat. Consistently, we saw a significant enrichment of BYac SNEPs in the region directly upstream rDNA (Figure 2B). Other examples are Ty retrotransposons. They differ greatly between natural strains, their epigenetic effect on nearby gene expression has long been observed [30] and their active LTR promoters are known to recruit the SAGA histone acetyltransferase [31]. Thus, nucleosomes residing near a Ty element in one strain but not in the other may harbor acetylation epi-polymorphisms. Consistently, BYac SNEPs were significantly enriched near BY Ty insertions (Figure 2B). If such large position effects were the general source of H3K14ac epipolymorphisms, one would expect SNEPs to cluster PLoS Genetics | www.plosgenetics.org 2 April 2010 | Volume 6 | Issue 4 | e1000913