alternative to irreversible nucleotide mutations. How BYac SNEPs accumulated at regulatory regions of conserved DNA is unclear. As mentioned above, it may occurred with the fixation of a trans- acting variation. Alternatively, accumulation may have occurred as a drift during laboratory culture conditions where fitness selection poorly applied. Future experiments examining a third wild strain will help determine if one of the two patterns is more ‘common’, if the stronger effect (acetylation fold-change) of BYac SNEP is peculiar, and if the abundance of SNEPs is similar in various pairwise comparisons of strains. What is the origin of this epigenomic variability? E. Richards proposed a classification of epigenotypes based on their depen- dency on DNA variation [4], where the obligatory, pure and facilitated qualifications relate to genetic controls that are full, absent or incomplete, respectively. Following this terminology, obligatory SNEPs may result from genetic factors acting in cis or in trans. Known cis-regulations are exemplified by position effects of transposable elements, rDNA repeats or telomeric sequences. Trans-acting genotypes may reside in histone acetyl-transferase or de-acetylase machineries, or in upstream regulatory factors. Such obligatory SNEPs could have been fixed together with their genetic determinants. In contrast, if some SNEPs are pure (independent of genotype) they likely result from their direct selection. As SNEPs seem to relate to the dynamics rather than the steady-state levels of gene expression, this selection may act through the ability to respond to environmental changes (the Baldwin effect). Also, interactions between epigenotypes and genotypes are expected since histone acetylation can modulate the buffering of cryptic genetic variations [42,43]. Acetylation of Lysine 14 of histone H3 at the beginning of protein-coding sequences has unambiguously been associated to high transcriptional activity in several studies [7,27,34]. It is therefore surprising that a preferential acetylation in one strain is not accompanied by a higher gene expression. This illustrates the complexity by which the various layers of inter-strain molecular differences are connected. Previous studies showed that DNA polymorphisms act on transcripts abundance in a complex manner [20], with a large extent of gene x environment effects [38] and that this genetic control was largely distinct from the control of protein levels [44]. Our results show that chromatin histone-borne modifications provide yet another layer of diversity, with non- trivial connections to genotypes and transcripts levels. SNEP identification and characterization provide a basis for population epigenetics of histone-borne modifications, and future quantitative epigenetics studies such as previously suggested [45,46] will define the nature of these dependencies, and their relevance to the control of complex traits. The abundance of SNEPs in highly-responsive genes and our observation that one SNEP correlated with the dynamics of gene activation upon stimulation suggest a contribution to gene x environment interactions. This is in full agreement with a previous report describing the contribution of H3K27me3 at the FLC locus of Arabidopsis to natural variation in cold-induced acceleration of flowering [17]. Except in such rare cases, gene-by-environment interactions have only been studied in the context of DNA variation. Integrating epigenotyping of histone marks in these investigations will likely better explain how individuals differ in their response to environmental changes. In particular, attempts to predict and optimize the response to specific treatments is at the heart of personalized medicine. Chemical inhibitors of histone deacetylase are used in anti-cancer therapies and seem promising to fight other diseases [47], and ChIP-SEQ technologies [9] will soon provide clinicians with epigenotyping possibilities. Our results suggest that histone modification profiles of Single Nucleosome Epi-Polymorphisms human individuals may greatly differ, with likely consequences on treatment outcome. Methods Nucleosomal DNA extraction and ChIP–CHIP Yeast strains used were BY4716 (MATalpha, laboratory [48]) and RM11-1a (MATa, derived from wild isolate [20]). We processed six BY and six RM independent cultures for H3K14ac ChIP, plus three BY and three RM independent cultures for nucleosome mapping, totalling 18 microarray hybridizations. Cells were grown to exponential phase in synthetic medium with 2% glucose (SDall) as in Brem et al. [20]. We followed the protocol of Liu et al. [7] for both nucleosomal DNA isolation and ChIP, except that incubation time with micrococcal nuclease (Worthington Biochemical) prior to immunopurification was increased to 30 min at 37uC to obtain mononucleosomes. ChIP was performed using 3 ml of anti-H3K14Ac polyclonal antibody (Upstate, 07–353). For H3K14ac, efficiency was controlled by quantifying acetylation at the MAT locus by real-time quantitative PCR. This locus, as opposed to the silenced HML and HMR loci, is acetylated [49] and since BY and RM have opposite signs, we expect ChIP to be enriched for HMLalpha1 sequence in the case of BY and HMRa1 sequence in the case of RM (Figure S11). Real-time quantitative PCR was performed on a LightCycler 1.5 (Roche) using FastStart DNA Master Plus SYBR GREEN I kit (Roche). Primer pairs were 59- AAATGTCTTGTCTTCTCTGCTC-39 and 59-ACTGTT- GCGCGAAGTAGT-39 for HMLalpha1 and 59-AAGAGCCC- AAAGGGAAAATC-39 and 59-AGGCTTTGCTTTCTTCTA-39 for HMRa1. ChIP and non-immunoprecipitated DNA fragments were linearly amplified using T-7 based in-vitro transcription as described previously [7] with few modifications. Briefly, the reaction mixture of 28.5 ml contained 18 ml template DNA, 5.2 ml 5x TdT buffer (Roche), 0.68 mM CoCl2 (Roche), 4.2 mM dTTP, 0.36 mM ddCTP, and 40 U terminal transferase (NEB). It was incubated at 37uC for 20 minutes and then stopped by adding 5 ml of 0.5 M EDTA (pH 8.0). Products were purified using Qiagen MinElute reaction cleanup kit and eluted in 20 ml nuclease free water, then concentrated to a 8 ml volume by Speed Vacuum centrifugation. The following were added: 0.6 ml of 25 mM T7- A18B primer, 1 ml of NEB buffer (2) and 0.4 ml of 5 mM dNTPs and the following thermal cycles were applied; 94uC for 2 min, decreasing to 35uC at -1uC/sec, hold down at 35uC for 2 min and decreasing to 25uC at -0.5uC/sec. Immediately after, 0.4 ml of Klenow enzyme (NEB) were added to the samples which were incubated at 37uC for 90 min. The reaction was halted by adding 5 ml of 0.5 M EDTA (pH 8.0). Products were purified using Qiagen MinElute reaction cleanup kit and eluted in 20 ml nuclease free water. The eluted samples were concentrated to a final volume of 5 ml. The IVT reaction mixture contained 5 ml nuclease free water, 2 ml 10X reaction buffer and 2 ml enzyme mix of MEGAshortscriptH T7 kit (Ambion), 6 ml Labeling NTP mix from Affymetrix, and 5 ml T-7 tailed DNA and incubated at 37uC for 16 hrs. Amplified RNAs were purified using RNeasy Mini kit (Qiagen) and eluted in 50 ml of nuclease free water. RNAs ($15 mg) were hybridized to GeneChip S. cerevisiae Tiling Array from Affymetrix [23] following manufac- turer protocol. Heat shock An isolated colony was picked to inoculate 4 ml SDall medium and incubated at 30uC with 220 rpm shaking for 12 to 16 h. This culture was used as a starter to inoculate 2 ml SDall medium at 0.1 OD600, which was grown for 6 hrs at 30uC with shaking. 1.5 ml of culture were then transferred to a microcentrifuge tube, incubated PLoS Genetics | www.plosgenetics.org 8 April 2010 | Volume 6 | Issue 4 | e1000913