Single Nucleosome Epi-Polymorphisms Figure 1. Nucleosome positioning in two unrelated natural S. cerevisiae strains. (A) Example of raw signals and nucleosome positioning inference in the region of the PER1 gene. Nucleosomal DNA was purified from each strains in triplicate, amplified linearly and hybridized to whole genome oligonucleotide Tiling arrays. Data were log-transformed and normalized using the quantile-quantile method and averaged across replicates to produce the probe-level signal intensities shown on the top panels. A Hidden Markov Model (HMM) similar to the one previously described [25] was applied to each strain independently to infer nucleosomal positioning (blue rectangles). Faded and plain colors represent ‘delocalized’ and ‘well-positioned’ nucleosomes, respectively, as defined previously [24]. Signal intensities are colored according to the HMM posterior probability to be within a nucleosome (cumulating delocalized and well-positioned). Nucleosome positions from the published atlas of Lee et al. [24], who used a strain isogenic to BY, are indicated by green rectangles and are also faded when reported as ‘delocalized’. (B) Genes (rows) were clustered based on profiles of nucleosome occupancy at their promoter in the BY strain (see Methods). Their order was then used to plot heatmaps of nucleosome occupancy around transcriptional start site in BY and RM, respectively, as well as expression divergence between the two strains (according to statistical significance at FDR 5% from the dataset of Brem et al. [20]). Left curves represent mean occupancy profiles of the six main classes of promoters. (C) Absence of correlation between promoter occupancy and expression divergence. Each dot represents one gene. X-axis: inter-strain difference in expression measured as log2(RM/BY) from Brem et al. [20]. Y-axis: inter-strain dissimilarity of promoter occupancy profiles. For each promoter region, the RM/BY dissimilarity was estimated as 1 - R, where R is the Spearman correlation coefficient between the BY and RM occupancy profiles shown in (B). r: Spearman correlation between the resulting X and Y data. doi:10.1371/journal.pgen.1000913.g001 together at particular hot spots. We clearly observed local correlations, as epipolymorphisms were 7 times more frequent than expected by chance among nucleosomes adjacent to SNEPs, and this effect could span over 10 nucleosomes upstream and downstream of a SNEP (Figure 2C). However, the majority (55%) of SNEPs were limited to a single nucleosome. This is unlikely to be a detection limitation, as 994 SNEPs had both flanking nucleosomes still scoring non-polymorphic at P,0.01. Thus, local correlation seems to be limited and epi-polymorphisms are frequently distributed on specific nucleosomes. SNEPs were not uniformly distributed along genes. The averaged H3K14 acetylation profile of both strains was consistent with previous descriptions [7,27], with enrichment downstream transcription start sites and decreased acetylation at the end of transcribed sequences (Figure 2D). However, strikingly, BYac SNEPs were abundant upstream TSS and around TES, while RMac SNEPs marked the second half of transcribed regions. These patterns were also visible when selecting only SNEPs with strong effect (.1.4-fold acetylation difference). This could result from a better recruitment of Rpd3S deacetylase behind elongating RNA polymerase II [32], as signs of elongation impairments were previously seen in RM [33]. It is important to note that this pattern of SNEP distribution reflects an average tendency, and that several genes present a totally different epigenetic pattern. For example, the NDE2 gene did not have BYac SNEP in promoter nor in terminator region, but had RMac SNEPs at the beginning of its coding region (Figure S5). Finally, RMac SNEPs were slightly more frequent than BYac SNEPs (58.5% versus 41.5%). SNEPs are not correlated to differential transcription levels Since acetylation of H3K14 is known to be associated with high transcription levels [7,27,34], its inter-strain variability could simply reflect inter-strain differences in gene expression. Transcriptional PLoS Genetics | www.plosgenetics.org 3 April 2010 | Volume 6 | Issue 4 | e1000913