This study reveals that H3K14ac epi-polymorphisms are not equally sensitive to environmental reprogramming. Some of them can be lost after temporary perturbations while others persist. This persistence clearly correlates with the presence of genetic determinants that encode epi-polymorphisms in the DNA. This genetic control is complex and resembles architectures previously described for eQTLs, with both cis and trans regulators and the presence of master regulators affecting numerous targets.
Importantly, our results further highlight the quantitative nature of the variation of acetylation levels. At any given time, a nucleosome of a given cell is or is not acetylated. Thus, acetylation is sometimes considered a discrete variable. However, the average acetylation level of this nucleosome across a population of cells is quantitative, because it depends on the number of cells that carry the acetylation mark, which corresponds to an equilibrium state of the population resulting from many biochemical reactions. The fact that this level varies as a complex trait shows that aceQTLs change the proportion of cells that are acetylated at their target nucleosome. In other words, aceQTLs are genotypes that modify the probability that a given nucleosome is acetylated in a given cell at a given time. How this happens will probably remain unknown until new technologies are developped to interrogate single nucleosome states in single cells. Also, the quantitative variability studied here is different from several epimutations described in plants where strong silencing of large chromosomal domains is established by a combination of many molecular and structural changes.
Whether the genetic control of chromatin variability also controls the level of nearby gene expression appears to be context specific. In humans, Gibbs et al.
did not find any consistent overlap between expression Quantitative Trait Loci (e
QTL) modifying the transcriptome of brain tissues and Quantitative Trait Loci modifying the methylome of these tissues (meth
. In contrast, Bell et al.
reported a clear consistency between e
QTL and meth
QTL in HapMap lymphoblastoid cell lines 
. Here we observed that about 70% of ace
QTLs linkages overlap with e
QTLs. The remaining fraction of ace
QTLs could correspond to regulations of non-coding transcripts, which were not interrogated by our study. Alternatively, given our previous association between SNEPs and transcriptional plasticity 
, it is possible that some ace
QTLs modify the chromatin in a way that manifests only upon transcriptional stimulation. In other words, genetic modifiers could increase K14 acetylation of a locus, which would then become more responsive to transcriptional activation or repression upon specific conditions. In such cases, ace
QTLs could participate in gene x environment interactions by creating epi-polymorphisms that personalize the way the genome responds to the environment.
We proved that the MAT
locus affects chromatin acetylation of many target loci. This locus determines the cell's mating type by dictating specific transcriptional programs. The MATα
allele encodes two regulatory proteins: α1, which activates α-specific genes, and α2, which represses expression of a
-specific genes. The MATa
allele encodes the a1 protein only, which heterodimerizes with α2 in diploid a
cells to form a repressor of haploid-specific genes. How specific transcriptional programs are established in a
cells has been the focus of many studies, revealing the interplay with chromatin acetylation regulation at specific target promoters. In α
cells, the cooperative binding of α2 and Mcm1 recruits the Tup1-Ssn6 repressor, which is known to interact with several histone deacetylases 
. In a
cells, α-specific genes are occupied by Sum1 which is known to recruit the NAD+-dependent histone deacetylase Hst1 to repress transcription 
. In our study, some loci controlled by MAT
displayed an epigenomic profile totally predictable given the known transcriptional control. This was the case for the BAR1
gene for example, which encodes a secreted protease specifically expressed in a
cells. The chromatin signature of the entire locus was affected by the mating type. MATa
strains displayed occupancy and acetylation intensities typical of highly expressed genes 
, with a marked nucleosome-free region near the transcription start site, and a high and low level of H3K14 acetylation in the first and second half of the coding region, respectively (Figure S5
). However, other loci controlled by MAT
displayed unexpected patterns of chromatin variation. One such example was the KAR4
locus, which encodes two forms of a transcription factor essential for nuclear fusion during mating. The long form is expressed in mitotically growing cells, and the short form is induced in response to pheromone from a transcriptional site about 30 nucleotides downstream the first ATG 
. Our study revealed marked differences between MATa
growing cells in the 3′ part of the gene, which were not accompanied by differential transcriptional levels (). How a
cells maintain elevated H3K14 acetylation on two nucleosomes at the end of the KAR4
coding region remains to be identified. It is possible that a
and α cells do not use the same strategy to maintain the locus transcriptionally active and responsive to pheromone. Comparing Ste12, Tup1, or Sum1 occupancy between a
and α cells might reveal some differences in this region. Alternatively, DNA replication initiated downstream KAR4
, at the ARS304 site, could have an effect if its timing differs between a
and α cells 
. Another particular case of mating-type specific chromatin organization was the promoter of the SAG1
gene, which encodes the α-agglutinin specifically expressed in α cells. The repressed state of a
cells corresponded to nucleosome occupancy downstream the TSS, and to hypoacetylation of H3K14 specifically at the -1 nucleosome (Figure S6
). These three examples illustrate that the mechanism by which MAT
alleles affect chromatin signatures at target genes is not simple: it can affect an entire locus (BAR1
), or a specific set of nucleosomes in the promoter (SAG1
) or the 3′ region (KAR4
More generally, the fact that ace
QTLs were not preferentially found at sites coding for chromatin modifying enzymes may seem counterintuitive: one could expect that DNA polymorphisms affect chromatin states by modifying the sequences of enzymes directly involved in chromatin regulation. However, protein complexes that regulate chromatin are themselves highly regulated, and any DNA polymorphism affecting these upstream regulators has the potential to induce chromatin modification indirectly. In fact, this is what happens with MAT
alleles: they do not code for chromatin remodelling enzymes but they determine distinct recruitments of chromatin modifiers at specific sites. This observation is very similar to results from eQTL mapping, from which we know that genetic modifiers of gene expression do not necessarily reside in direct transcriptional regulators 
. For example, the AMN1, GPA1, IRA2
yeast genes were all validated as e
QTL players but they do not encode direct regulators of transcription 
. These polymorphisms affect gene expression by perturbing regulatory networks upstream of transcriptional machineries. The results presented here suggest that ace
QTLs likely follow a similar rule: causative polymorphisms may reside not only within chromatin modifying complexes but also in their upstream regulators.
We show that a transient environmental change imposed by TSA treatment can reprogram a subset of H3K14ac epi-polymorphisms: numerous new SNEPs were induced, and numerous initial SNEPs were lost. An important consideration is that TSA imposed a perturbation but did not necessarily saturate the acetylation of H3K14 on all nucleosomes. In normal conditions, H3K14 acetylation levels result from a balance between the activity of histone acetyltransferases (HATs) and deacetylases (HDACs). In S. cerevisiae
, at least three HATs are known to acetylate Lysine 14 of Histone H3: Gcn5p 
, Sas3p 
, and Hpa2p 
, and deacetylation of Lysine 14 can be attributed to HDACs of all three classes: Hos3p and Rpd3p of class I 
, Hda1p of class II 
and Sir2p of class III 
. TSA is known to induce a bulk hyperacetylation by inhibiting the activity of a subset of these HDACs: while Rpd3p and Hda1p are sensitive, Hos3p and Sir2p remain active. Thus, the perturbation applied in our experiment did not necessarily saturate K14 acetylation on the entire chromatin. In addition to the direct effect of TSA on HDACs that deacetylate H3K14, the treatment may have perturbed this lysine residue indirectly. The very slow growth in presence of TSA (not shown) suggests that cells profoundly reshaped molecular profiles during treatment, with possible consequences on the regulations of HATs and HDACs.
The reprogramming observed preferentially corresponded to a gain of acetylation in the BY strain, with a majority of labile SNEPs corresponding to hypo-acetylated nucleosomes in the BY strain that returned to levels comparable to those of the RM strain. An intuitive interpretation of this asymmetry would be that TSA was more efficient to induce hyperacetylation in BY than in RM. The strains are probably not equally sensitive to TSA, given the two previously mapped QTLs of growth fitness in the presence of TSA that segregate in the BYxRM cross 
. However, the possibility that BY suffered a more pronounced hyperacetylation does not explain why only a subset of nucleosomes were preferentially reprogrammed. Alternatively, the strains may differ in their recovering efficiency. Although after 20 generations all HDAC complexes are young enough to consider they never bound the chemical inhibitor, it is still possible that the chromatin of the BY strain did not fully return to equilibrium. Then again, why would an incomplete recovery target preferentially a subset of nucleosomes? Our observation that the nucleosomes affected are largely those initially hypoacetylated suggests a third and complementary interpretation: the BY strain may have accumulated hypoacetylation ‘epimutations’ that were cured by the treatment. BY is a strain that has been maintained in laboratories for decades and is known to possess many deleterious mutations that would likely be counter-selected in the wild. Our results raise the possibility that it has also drifted at the epigenetic level, and it will be very exciting to test this hypothesis in future experiments.
More generally, it will be essential to question the origin of the ‘labile’ SNEPs: those which gained but also those which lost acetylation in BY, and the few where the change happened in RM. Theoretically, the differences in these epigenotypes may have occurred any time between the initial divergence of the strains and the last hours before the stocks were frozen in our laboratory. In other words, our study identified their lability but not their origin and age. A related question is how stable are labile and newly induced SNEPs: how harsh a treatment is needed to reprogram them? If some ‘labile’ SNEPs are old, they have been maintained for a long time and one would expect them to be stable unless extreme environmental perturbations are experienced, like in our TSA-based assay. Likewise, it is possible that additional SNEPs could have been modified if we had applied a stronger or longer treatment. As mentioned above, class III HDACs such as Sir2p are not inhibited by TSA, and other SNEPs would probably be called ‘labile’ if an inhibitor of sirtuins was used instead of TSA. In contrast, some ‘labile’ SNEPs may be very unstable and might also disappear after a prolonged but unperturbed culture. It will therefore be interesting to monitor the dynamics of SNEP appearance and loss in unperturbed conditions. A time-course experiment tracking the H3K14ac epigenome of one strain over long culture periods would help determine its stability.
How and for how long were new SNEPs induced despite the fact that the treatment applied was the same for the two strains? As mentioned above, this can possibly result from a difference in the way the strains respond to the treatment, and this difference might or not be genetically encoded. Although our experiments were not designed to address this, it is also possible that epi-polymorphisms arise stochastically in particular environments regardless of the genetic background. This has been suggested by a recent study where the methylome of isogenic mice fed with high levels of methyl precursors was tracked over generations 
. This treatment was shown to increase inter-individual epigenome diversity, although the diet itself was common to all animals. Thus, induced epi-polymorphisms may reflect not only differences in the history of past environmental exposures, but also genetic or stochastic differences in the way individuals reprogram their epigenome in response to specific environments.
We observed a clear correlation between environmental persistence and genetic control of acetylation variation. Importantly, the two datasets (reprogramming and QTL mapping) were generated and analysed independently: at different dates, by different experimenters, the former using the parental strains only and the latter using the segregants. Thus, we believe that this correlation truly reflects the robustness of DNA-encoded epi-polymorphisms to environmental reprogramming. However, our observations do not imply that all cases of epi-polymorphism persistence result from their anchoring in DNA. It remains entirely possible that specific cases have a purely epigenetic basis. For example, H3K14 acetylation may be more robust to environmental perturbation if it is accompanied by additional epigenetic marks that are commonly associated with it, such as H3K4 di- or tri-methylation, or H3K9 acetylation 
. If such marks drive H3K14 acetylation and are not affected by the environmental change, then persistence is ensured without a DNA-encoded control.
Given our observation that both labile and persistent epi-polymorphisms coexist abundantly in natural epigenomes, we emphasize the importance of the stability of epi-polymorphism in the current debate on whether and how epigenotypes contribute to evolutionary mechanisms. As outlined by B. Turner, this question is fundamental because epi-polymorphisms potentially enable environmental conditions to reprogram molecular events for a durable time. This way, “epigenetic processes might contribute to evolutionary change, at least in part by expanding the range of phenotypic variants on which natural selection can act
. A key factor for selection to act is then the amount of time during which the ‘novel’ phenotypic variants (those generated by chromatin changes) are exposed. If too short, individuals with beneficial traits may not have time to expand in the population, especially if the phenotypic variants consist of small quantitative differences. Although our results did not link SNEPs to phenotypic traits, they suggest that the amount of time for selection to act may differ if the phenotypic variants result from labile or from persistent epi-polymorphisms (). This duration depends on the stability of the new epigenotype and on the probability to encounter environmental conditions that change its state. If the epigenotype is robust to environmental perturbations, then the phenotype is exposed as long as other genetic or epigenetic modifiers of it are acquired. Natural selection is therefore more likely to act on phenotypic variants resulting from persistent epi-polymorphisms. Note that such high persistence can sometimes result from a full genetic control. In this case, the fact that epi-polymorphisms are involved no longer matters: selection acts on the genetic determinant regardless of the mechanism leading to the phenotype.
Distinct evolutionary implication of labile and persistent epi-polymorphisms.
Importantly, loci harboring DNA-encoded epi-polymorphisms may remain highly susceptible to epigenetic regulations: as mentioned above, SNEPs represent small quantitative differences of molecular regulations, and it is likely that they do not prevent from switching between radically different epigenetic states. Thus, the prolonged duration of DNA-encoded epi-polymorphisms does not necessarily impair fitness in fluctuating environments, where adaptation requires rapid and profound chromatin remodelling at critical loci.
In addition, chromatin changes may reveal the effect of cryptic genetic variations. For example, a mutation occuring at a silenced locus can remain cryptic until silencing of the locus is alleviated. Such epigenetic alleviation might also be either labile or persistent, with different consequences on the cryptic variation: the phenotype and therefore the cryptic variation itself may be exposed to selection for a longer period of time if alleviation persists.
Altogether, our observations provide a necessary basis for the upcoming development of population epigenetics, where epi-polymorphisms of natural populations will be interpreted and possibly associated to the variation of common traits.