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Title: Conserved Substitution Patterns around Nucleosome Footprints in Eukaryotes and Archaea Derive from Frequent Nucleosome Repositioning through Evolution}
Article:
Abstract:
Nucleosomes, the basic repeat units of eukaryotic chromatin, have been suggested to influence the evolution of eukaryotic genomes, both by altering the propensity of DNA to mutate and by selection acting to maintain or exclude nucleosomes in particular locations. Contrary to the popular idea that nucleosomes are unique to eukaryotes, histone proteins have also been discovered in some archaeal genomes. Archaeal nucleosomes, however, are quite unlike their eukaryotic counterparts in many respects, including their assembly into tetramers( rather than octamers) from histone proteins that lack N- and C-terminal tails. Here, we show that despite these fundamental differences the association between nucleosome footprints and sequence evolution is strikingly conserved between humans and the model archaeon Haloferax volcanii. In light of this finding we examine whether selection or mutation can explain concordant substitution patterns in the two kingdoms. Unexpectedly, we find that neither the mutation nor the selection model are sufficient to explain the observed association between nucleosomes and sequence divergence. Instead, we demonstrate that nucleosome-associated substitution patterns are more consistent with a third model where sequence divergence results in frequent repositioning of nucleosomes during evolution. Indeed, we show that nucleosome repositioning is both necessary and largely sufficient to explain the association between current nucleosome positions and biased substitution patterns. This finding highlights the importance of considering the direction of causality between genetic and epigenetic change.
Introduction:
Both in vitro and in vivo, nucleosomes are non-randomly positioned with regard to the underlying sequence, forming preferentially on stretches of DNA that – by virtue of their sequence composition – are more amenable to being wrapped around the histone core[1]–[3]. Consequently, changes at the sequence level during evolution can bring about changes in nucleosome positioning and occupancy[4],[5], providing a simple example of how genetic changes can locally alter epigenetic states. Conversely, epigenetic states – here used in the broadest sense to include nucleosome positions, histone marks, DNA methylation state, etc. – can influence evolution at the sequence level. As applied to nucleosomes, three broad mechanisms can be distinguished. First, the presence of nucleosomes can affect the efficacy of DNA repair by altering the structural context in which lesions need to be detected and removed[6]. Second, rates of initial lesion formation can vary as a function of nucleosome occupancy. For example, a recent mutation accumulation experiment in yeast revealed a reduced incidence of C:G to T:A changes in nucleosome-bound regions[7], consistent with a model where DNA, when wound around a protein, is less likely to expose cytosine residues to conditions that promote spontaneous deamination. Third, since nucleosome positioning can mediate access to promoter elements or transcription factor binding sites[8], selection may eliminate mutations that alter nucleosome position in ways that disrupt proper access to these functionally important sites. In short, nucleosomes can affect evolution at the sequence level by modulating mutation and repair dynamics( thereby biasing the emergence of novel variants) and by exerting selective pressure on the underlying sequence( thereby altering fixation probabilities). Multiple recent studies have claimed support for either biased mutation or biased selection as the underlying cause behind( often strikingly) uneven divergence patterns around nucleosome in various eukaryotes including human, yeast, and C. elegans[7],[9]–[14]. For example, A:T to G:C substitutions were found to be more common closer to the nucleosome mid-point in humans, whereas C:G to T:A changes were enriched outside the nucleosome[9]. Interpretation of these trends has up to now proceeded from the assumption that experimentally determined nucleosome positions correspond closely to ancestral positions and that, as a result, current positions are informative about the chromatin context in which substitutions occurred. However, if this assumption is wrong and nucleosomes are repositioned following a change at the sequence level, conclusions about the relationship between nucleotide substitutions and nucleosome positions might change dramatically( Fig. 1). Might A:T to G:C substitutions, for example, be more common near the nucleosome dyad( the centre position of the binding footprint) simply because such a substitution tends, on average, to attract rather than repel nucleosomes? Here, in an effort to disentangle cause and effect in the relationship between nucleosome binding and sequence evolution, we compare substitution patterns around nucleosome footprints in humans to substitution dynamics in the Haloferax clade, a group of halophilic archaea that includes the model haloarchaeon Haloferax volcanii, for which nucleosome organization was recently determined at high resolution[15]. Like their orthologs in eukaryotes, archaeal histones form multimeric complexes that preferentially assemble onto more bendable DNA templates[16],[17], with sequences bound in vivo exhibiting a higher average GC content in both eukaryotes and archaea[15],[18]. Further, nucleosome organization around Hfx. volcanii promoters is strongly reminiscent of nucleosome architecture in eukaryotes[15] suggesting that archaeal nucleosomes play similar roles in regulating access to DNA and controlling gene expression. Importantly, however, archaeal nucleosomes also differ in multiple respects from their eukaryotic counterparts: notably, although archaeal histones assemble into tetramers, homologous to the( H3–H4) 2 tetramers seen in eukaryotes, they do not form octamers[19]. Consequently, archaeal nucleosomes are smaller and wrap less DNA, ∼85 nucleotides( nt)[17] compared to ∼147 nt in eukaryotes, with shorter linkers separating consecutive nucleosomes[15]. In addition, whereas eukaryotic histones sport N- and C-terminal tails, which can be acetylated, methylated or otherwise modified to generate distinct chromatin states, archaeal histones lack pronounced tails and there is currently no evidence for their post-translational modification[20]. Here, we demonstrate that – despite such differences in nucleosome structure, global nucleotide substitution profiles and general cellular physiology – nucleosome-associated substitution patterns along the Hfx. volcanii lineage are remarkably similar to those observed in humans( as well as in Drosophila melanogaster). In particular, strong similarities exist regarding which base-specific changes are more and which are less commonly observed when approaching the nucleosome dyad. We go on to show that these dyad-oriented trends break down in the respective sister lineages, an observation that is neither consistent with mutation nor purifying selection acting on nucleosomes that are positionally static over evolutionary time. Instead, we show that these patterns are consistent with widespread local repositioning of nucleosomes in response to substitutions. Our analysis provides a powerful caveat that the causal link between genetic and epigenetic change must be considered when assessing selection and mutation biases in the context of chromatin architecture.
Discussion:
Our analysis revealed that substitution patterns in and around nucleosome footprints are remarkably similar along the Hfx. volcanii and human branches, with substitution rates consistently reduced for some( e. g. C:G to T:A) but elevated for other( e. g. A:T to G:C) base changes when approaching the dyad. These shared biases are observed despite radical differences in global substitution dynamics and structural differences between the histone complexes involved. Exploring whether common factors generate these signatures in both clades, we discovered that substitution trends in the respective sister lineages do not show the same behavior, an observation that is inconsistent with mutational biases or purifying selection acting on nucleosomes that are positionally inert through evolution. On the contrary, our analysis demonstrates that many nucleosomes must have repositioned from their ancestral locations. Further, we argue that biased repositioning is largely sufficient to explain the association between nucleosome topology and nucleotide substitution rates, whereby nucleosomes shift( likely locally) from their ancestral position in response to a change in the underlying sequence, and do so in line with known histone binding preferences. The results presented here also highlight that defining a unique dyad, rather than considering a partly overlapping, probabilistic ensemble of nucleosome footprints, might lead to misleading conclusions because sequence changes – while not affecting the local ensemble of positions per se – can affect which translational positions are occupied more frequently and hence affect which of these positions is picked when calling the dyad. Our findings do not imply that mutations are not modulated by nucleosome occupancy or that selection does not act to maintain at least a subset of nucleosomes in functionally relevant positions. In fact, recent results from mutation accumulation lines in yeast[7] strongly support the notion that there are systematic differences in mutation rates for sequences bound by nucleosomes versus linker DNA. A role for mutation and/or selection also appears supported by the biased incidence of single nucleotide polymorphisms around nucleosomes in human and yeast[12],[14],[24],[25], although the possible effect of different ancestral nucleosome positions was not considered in these studies. What our findings do suggest, however, is that mutation/selection biases are not sufficient to explain the observed association between nucleosome positions and nucleotide substitution patterns between species and must operate, if they do, on a background of nucleosome repositioning. As a result, our analysis provides an important general caveat to interpreting substitution dynamics in relation to observable epigenetic states because these states might have been different at the time when the substitutions occurred. Future analyses, especially when concerned with detecting signatures of purifying and, above all, positive selection, should take nucleosome mobility into account and ideally model explicitly how mutation biases, selection, and re-positioning interact to determine the co-evolution of nucleosome positions and the underlying sequence. Such studies should focus on closely related sister taxa as – with increasing evolutionary distance – the causal relationship between genetic and nucleosome positional change will become increasingly harder to decipher. This is principally because multiple nucleotide changes, which may affect binding in a hard-to-predict combinatorial fashion, need to be considered concurrently. Indeed, comparing S. cerevisiae and S. paradoxus, which are substantially further diverged than the primate, Drosophila and Haloferax sister pairs analyzed above, we do not recover analogous trends at 4-fold synonymous sites, despite the availability of high-resolution nucleosome datasets( Fig. S5). In addition, studies of the type conducted by Chen and colleagues[7], where ancestral footprints can be assayed directly and changes followed forward in time will be invaluable to learn more about mutation biases in vivo and understand, for example, whether mutational biases largely reinforce current positioning( as might be the case for reduced C:G to T:A rates inside of nucleosomes[7], where changes towards nucleosome-disfavouring, AT-rich sequence would be concentrated in already disfavouring sequence) or indirectly favour positional stability over the longer term by promoting compensatory mutation in the regions that assume a new mutation regime when the nucleosome is repositioned.
Genome sequences as well as epigenetic states , such as DNA methylation or nucleosome binding patterns , change during evolution . But what is the causal relationship between the two ? We already know that nucleotide variation within and between species is distributed unevenly around nucleosome footprints , but does this mean that sequence evolution follows a biased course because the presence of nucleosomes affects mutation and DNA repair dynamics ? Or is it , in fact , the other way around , i . e . changes happen at the DNA level and prompt shifts in nucleosome positioning ? To investigate the direction of causality in genetic versus epigenetic evolution , we analyze substitutions patterns in eukaryotes as well as the archaeon Haloferax volcanii in the context of genome-wide nucleosome binding maps . We demonstrate that the relationship between nucleosome positions and between-species divergence patterns , strikingly similar in eukaryotes and archaea , can be explained in large parts by nucleosomes shifting positions in response to substitution , although both mutation and selection biases might still exist . Our results illustrate that it is important to consider the direction of causality between epigenetic and genetic change when analyzing patterns of sequence divergence and using sequence conservation to infer selection on epigenetic states .