You will be presented with the abstract, introduction, and discussion section from a biomedical research article. Your task is to create a summary that a layperson can understand, capturing the essence of the research article. Keep essential scientific terms, but ensure that the language remains clear and the concepts are explained in an uncomplicated manner.

Title: Promoter Nucleosome Organization Shapes the Evolution of Gene Expression}
Article:
Abstract:
Understanding why genes evolve at different rates is fundamental to evolutionary thinking. In species of the budding yeast, the rate at which genes diverge in expression correlates with the organization of their promoter nucleosomes: genes lacking a nucleosome-free region( denoted OPN for “Occupied Proximal Nucleosomes”) vary widely between the species, while the expression of those containing NFR( denoted DPN for “Depleted Proximal Nucleosomes”) remains largely conserved. To examine if early evolutionary dynamics contributes to this difference in divergence, we artificially selected for high expression of GFP–fused proteins. Surprisingly, selection was equally successful for OPN and DPN genes, with ∼80% of genes in each group stably increasing in expression by a similar amount. Notably, the two groups adapted by distinct mechanisms: DPN–selected strains duplicated large genomic regions, while OPN–selected strains favored trans mutations not involving duplications. When selection was removed, DPN( but not OPN) genes reverted rapidly to wild-type expression levels, consistent with their lower diversity between species. Our results suggest that promoter organization constrains the early evolutionary dynamics and in this way biases the path of long-term evolution.
Introduction:
The plasticity of biological traits is manifested on multiple time scales. Regulatory mechanisms govern the physiological adaptation of an individual to changing conditions. On evolutionary time scales, phenotypes are modulated by genetic mutations. Although operating on very different time scales, regulatory variance and evolvability were proposed to be linked[1]–[8]. For example, a trait that needs to be buffered against environmental or stochastic variations will show a limited regulatory variance, and will be harder to perturb by genetic mutations. A related idea is that regulatory variance directs the evolutionary dynamics by marking the directions most susceptible to changes. Experimental evidences supporting these ideas are still sparse, but recent studies in yeast provided genome-wide support to this linkage in the context of gene expression. Adaptation of cells to different environmental conditions depends largely on changes in expression levels, whereas evolution depends on changes in both expression and function. While most studies of evolutionary changes focused on changes in gene function, the role of gene expression in generating phenotypic diversity was emphasized by experiments that traced phenotypic and morphological differences to variations in gene expression[9]–[12] and by genome-wide mapping of gene expression profiles which demonstrated rapid divergence even between closely related species[13]–[19]. In yeast, the divergence of gene expression was linked to the organization of promoter nucleosomes, thereby connecting evolutionary divergence with physiological regulation[20]–[22]. Genes whose expression diverged rapidly typically lack an NFR proximal to the transcription start site( OPN genes), while the expression of genes with a pronounced proximal NFR( DPN genes) remained largely conserved. OPN genes are additionally more responsive to environmental changes, display a higher cell-to-cell variability( noise) and tend to have a TATA box in their promoters[20]. Multiple, not mutually exclusive, processes can explain the increased divergence of OPN genes. First, the highly responsive OPN promoters may be more sensitive to mutations accumulating by random drift. This promoter organization may enhance sensitivity to cis mutation in the promoter itself due, for example to non-linear interactions between promoter nucleosomes and transcription factors. Similarly, the spectrum of effective trans mutations may be larger for OPN genes[22]. Indeed, OPN promoters integrate a larger number of signals, are more responsive to regulatory factors and diverge more in mutation-accumulation experiments where selection is eliminated[23]. A second possibility is the two classes of genes are subject to distinct selection forces. Increased expression of low-responsive( DPN) genes may be more deleterious and will therefore be eliminated more efficiently by purifying selection. Similarly, mutations in high-responsive genes may contribute more to evolutionary adaptation leading to their rapid fixation. Alternatively, DPN and OPN genes may be subject to similar selection forces for changing expression, but selection is more easily satisfied by OPN genes due to their flexible promoter structure. This last possibility would provide a direct support to the idea that flexible promoter organization can direct the dynamic path taken by evolution. To examine the hypothesis that OPN and DPN genes differ in the way by which they respond to identical selection forces, we artificially selected for high expression of GFP-fused yeast proteins and examined the genomic response to this selection. The expression of dozens of GFP-fused proteins was successfully increased, irrespectively of their promoter class. Notably, promoter class did influence the genetic change leading to the increased expression: Selection for high expression of DPN genes resulted in duplication of large genomic region( mostly full chromosomes) containing the gene of interest. In contrast, large-scale duplications were much less prevalent in OPN genes, which changed their expression primarily through trans mutations not involving duplications. When selection was removed, DPN( but not OPN) strains reverted back to wild-type expression levels, consistent with their lower diversity between species. Our results suggest that promoter organization impacts on the early evolutionary dynamics and by this biases the path of long-term evolution.
Discussion:
Genetic changes leading to increased gene expression can be classified as regulatory cis-effects, regulatory trans-effects and segmental duplications that increase gene's copy number. By regulatory cis-effects we refer to mutations in the close vicinity of the gene promoters, altering, for example, the binding of regulatory factors. Trans-effects refer to mutations that occur elsewhere in the genome, for example modulating the activity of an upstream transcription factor or signaling protein. Such mutations are expected to have a wider influence on gene expression compared to cis-effects as they will modulate the expression of many( or all) targets of the associated trans factor. Finally, gene expression can also be increased by duplication, consisting of either a full chromosome or a chromosomal region containing the gene of interest. Chromosome duplications will modulate the expression of hundreds of unrelated genes. Studies that compared gene expression between related organisms revealed that most expression changes result from regulatory cis and trans mutations, with cis-effects dominating the divergence between species, while trans-effects dominate the divergence between different strains of the same species[18],[28],[29]. At least in yeast, trans-effects preferentially modulate the expression of genes of the OPN promoter type, but are less significant at genes of the DPN class[18]. Large scale chromosomal duplications are typically not observed when comparing yeast strains and species. This distribution of effects could reflect the frequency of initial mutations arising in the population, the interplay between their selective advantages versus possible deleterious outcomes, or the probability of reverting back the original mutations. Our results provide a complementary view on the early response to selection for high expression. We find that the genetic changes dominating this initial adaptation differ from those dominating the long-term evolution. First, regulatory cis effects were not observed. Rather, expression was modulated either by large-scale duplications or by regulatory trans mutations. Most notably, the choice between trans mutations and large-scale duplications was essentially dictated by the gene promoter class: genes of the DPN class changed expression almost exclusively through duplications, whereas genes of the OPN class did so primarily through trans-effects not involving duplications. This difference between early and later evolutionary mechanisms reflects transition from ‘general’ to more specific solutions: for the DPN class, the general solution of large-scale duplications appears to be the most easily accessible. It arises easily, and is indeed frequently observed during initial selections[30]–[33]. This solution, however, is more difficult to maintain due to pleiotropic effects and the high frequency by which the additional chromosome can be lost, which may explain its absence in species or strains. For the OPN class, trans-effects appear to be the more accessible solution, while chromosome duplications are observed at significantly lower frequency. OPN genes occupy the same chromosomes as DPN genes and thus should have the same likelihood of being duplicated. The preferential modulation of OPN genes by trans effects may thus indicate the larger spectrum of trans factors that influence the expression of those genes which increases the number of potential trans mutations. Notably, we find that although trans mutations arising in our strains did decrease the competitive growth fitness to the same extent as did chromosome duplications, they were easier to maintain. We hypothesize that this reflects the large spectrum of mutations that can compensate for the reduction in cell growth, making it unlikely that the cells precisely revert the mutation leading to the increased expression. This is in contrast to the case of chromosomal duplication, where reversion of the original mutation( loss of the duplicated chromosome) is most likely. Notably, this difference in reversion strategy may explain the prevalence of trans-dependent divergence of OPN genes[18]. These trans mutations may have arisen during transient selection for high expression, but were maintained even after selection was removed, possibly due to a compensatory mutation. Consistent with this possibility, we recently demonstrated extensive trans-dependent expression variability of OPN genes that is buffered by the activity of chromatin regulators[34]. Finally, the most specific solutions( e. g. cis regulatory mutations) require more time to emerge compared to the other more general processes, yet their specificity to the gene of interest allows their long-term maintenance in the population which may explain their dominance in the divergence between species[18],[28],[35]. It may also be that those cis effects are mostly neutral and do not arise in response to selection, at least not in response to a strong selection. Indeed, if expression can be easily increased through trans mutations or duplication, those will arise quickly and will reduce further pressure for the emergence of cis mutation. It should be noted that our study was performed in a haploid background, which favors the emergence of recessive mutations. In contrast, in nature yeast cells exist primarily as diploids. Many of the trans mutations we identified will have no effect in a diploid background. Yet, a large fraction of them were only partially recessive and thereby manifested also in the diploid background. It is likely that those mutations will dominate the initial evolution of OPN genes also in a diploid background. In conclusion, we find that gene expression readily evolves in response to strong selection. Furthermore, we propose that the genetic mechanisms by which expression evolves, and hence the stability of this genetic change, depends on the organization of gene promoter. Together, our study supports the idea that regulatory variance shapes evolutionary path by biasing long-term evolutionary changes to genes with flexible OPN promoter organization.
Species diverge by mutations that change protein structure or protein regulation . While the evolution of protein sequence was studied extensively , much less is known about the divergence of gene expression . To better understand the process of gene expression evolution , we characterized the early genomic response of yeast cells to selection for high gene expression . Notably , the response to selection was strongly dependent on the organization of the gene promoter: genes whose promoters had a pronounced nucleosome-free region ( NFR ) primarily duplicated the chromosome containing the gene of interest , while genes whose promoters lacked a pronounced NFR adapted by trans mutations not involving duplications . Further , when selection was removed , the former ( but not the later ) evolved strains reverted rapidly to wild-type expression levels , consistent with their lower diversity between species . Together , our study provides strong support to the idea that physiological regulation impacts the evolutionary path and suggests that , by regulating promoter nucleosomes , cells can regulate the response to selection and control the long-term stability of the selected changes .