early in research on the molecular basis of phenotypic variation the focus was primarily on mutations in the coding regions ( exons ) of genes . but as first noted by king and wilson , substantial physiological differences can be seen between closely related species despite almost identical sets of proteins , and it is now generally accepted that distinctions between species are defined not only by their ensemble of genes but , critically , by how those genes are regulated . for example , dramatic differences in the body plan of related insects have been traced to differences in the expression of developmentally regulated genes [ 2 - 4 ] , and the classic example of variation in beak shape among darwin 's finches appears to be controlled by variation in expression levels of the gene encoding bmp4 . surveying 331 previously reported mutations underlying phenotypic changes , stern and orgogozo   found that approximately 22% were regulatory changes , and the proportion of documented regulatory changes is increasing annually and is even larger for inter - species differences . more recent studies using advanced technologies , including microarrays or high - throughput sequencing , have compared the genome - wide expression programs of related species [ 7 - 16 ] or strains [ 17 - 29 ] and revealed thousands of differences in the expression of orthologous genes . identifying the regulatory changes underlying specific expression differences has , however , been more difficult : little progress has been made in connecting expression divergence with regulatory sequence divergence , and the degree of sequence conservation at individual promoters and regulatory elements can not predict the degree of expression divergence of the associated genes [ 30 - 34 ] . what has emerged is a more general distinction : some genes have a much greater propensity to diverge in their expression than others . here we discuss recent studies in yeast on the promoter architectures underlying these differences , and how they may contribute to the evolvability of gene expression . yeast is an excellent model for studying the evolution of gene expression because of its simplicity as a unicellular organism with short and well - defined promoter regions , ease of genetic manipulation and a wealth of functional genomics data . the notion that there are two kinds of promoters in yeast , with different functional and architectural properties , was developed long ago by struhl and colleagues , who extensively studied the regulation of the adjacent yeast genes his3 and pet56 and suggested the presence of distinct core promoters that control constitutive versus inducible gene expression . more recent studies have shown that these distinctions correspond to distinct evolutionary properties : whereas the expression of some genes has diverged between related yeasts the expression of others has remained stable . notably , this gene - specific tendency is maintained in multiple studies comparing the genomic expression patterns of different yeasts . despite the fact that these studies were on different sets of yeast strains or species grown in different environments , and that different quantities ( expression levels or ratios ) were measured and different computational and experimental methods used , their results show significant correlations : genes whose expression diverged according to one study were often found to diverge in the other studies . moreover , these genes also preferentially diverged in expression in ' mutation accumulation ' experiments , where cells were allowed to accumulate mutations in conditions in which the effects of natural selection were minimized . thus , we believe that expression divergence of these genes in multiple datasets is not due to increased positive selection ( or relaxation of purifying selection ) , but instead reflects an inherent capacity for expression divergence . this capacity of a gene to evolve in expression can be quantified by measuring its ' expression divergence ' - that is , a mathematical quantification of how much the expression of a gene differs among evolutionarily related yeast species or strains . expression divergence correlates strongly with gene responsiveness , namely the extent by which a gene 's expression is altered by the environment , and with expression noise , namely the extent by which a gene 's expression differs among genetically identical cells . that is , genes whose expression is strongly regulated between different conditions display noisy expression and evolve rapidly between related strains or species . thus , it is possible that genes differ in their capacity for expression flexibility , which is manifested at various timescales : during evolution in response to mutations ; during physiological responses to environmental changes ; and within a population of cells as a result of stochastic fluctuations . the capacity for expression divergence ( or flexibility ) has been linked to several characteristics of gene promoters . the simplest association is with the number of binding sites for transcriptional regulators : promoters of flexible genes are characterized by a relatively large number of binding sites . this is perhaps not surprising , since the expression of genes with many regulators ( and binding sites ) can be affected by mutations in any one of these regulators ( or promoter binding sites ) , thus increasing their mutational target size - that is , the number of possible mutations that would affect the expression of these genes . one particular promoter binding site stands out for its large influence on expression divergence : promoters that contain a tata box show a remarkable increase in expression divergence , as well as in responsiveness and in noise . the distinction between genes with promoters containing a tata box and those without stands when the number of transcriptional regulators or of promoter binding sites is controlled ; it is also consistent among genes from different functional classes - for example , those encoding membrane proteins , genes encoding metabolic proteins , and genes encoding ribosomal proteins ( although these different groups also differ widely in the proportion of genes with promoters containing tata boxes ) . strikingly , increased expression divergence of tata - containing genes has been observed in species ranging from yeast to mammals , including also mutation - accumulation lines of yeasts , flies and worms , suggesting that it reflects a general phenomenon . interestingly , the promoters of tata - containing genes are not associated with more mutations but only with increased expression divergence . thus , we propose that promoters carrying a tata box are inherently more sensitive to genetic perturbations than tata - less promoters . this is also consistent with the distinction between constitutive and inducible genes and with previous studies that demonstrated that a canonical tata box is important for dynamic regulation of gene expression whereas other sequence elements are important for maintaining constitutive expression levels . the tata box is a ubiquitous core promoter element that is bound by the transcription pre - initiation complex ( pic ) . transcription can be considered as a two - step process : first the pic is recruited by transcription factors and assembles at the core promoter together with rna polymerase ; and second , the polymerase is released from the pic and transcribes the gene . the second step can be repeated multiple times ( re - initiation ) if the pic remains bound to the core promoter , and this is believed to be facilitated by the tata box [ 42 - 44 ] . thus , a tata box could increase the extent of re - initiation , thereby amplifying gene expression . notably , the binding of the pic to the tata box and the binding of transcription factors to other sites could be cooperative . this would make the effect of the tata box on gene expression nonlinear , as any amplification of transcription factor binding would stabilize pic binding and cause a further increase in re - initiation . in this way , tata - containing genes could be more sensitive to regulatory mutations than tata - less genes . importantly , tata - containing promoters differ from other promoters not only in their expression flexibility but also in other properties , and so it is possible that these secondary characteristics underlie their increased expression flexibility . perhaps the most notable feature of tata promoters is their atypical chromatin structure [ 46 - 48 ] . at most yeast promoters , the region directly upstream of the transcription start site contains transcription factor binding sites and is nucleosome - free , increasing the accessibility of the binding sites to transcriptional regulators   ( figure 1 ) . by contrast , at promoters with high expression flexibility , and at those containing a tata - box , this region tends to be more occupied by nucleosomes ( figure 1 ) . we and others have proposed that because nucleo somes are thought to interfere with the binding of regulatory proteins , the regulation of nucleosome states might fine tune the expression of these genes [ 46 - 48,50 ] . such increased dependence on the regulation of chromatin structure is indeed observed : promoters that are relatively more occupied by nucleosomes show relatively large changes in expression when genes encoding chromatin regulators are mutated or deleted . as with the effect of the number of transcription factors , an increased dependence on chromatin regulators increases the mutational target size , affecting expression of these genes . any mutation in a gene encoding a relevant chromatin regulator , or an upstream gene regulating the activity of the chromatin regulator , could affect transcription of the downstream target gene . top : the architecture of a typical promoter in which nucleosomes are regularly positioned but are excluded from a particular region upstream of the transcription start site . this nucleosome - free region ( nfr ) contains accessible binding sites for ( few ) transcriptional regulators ( tf ) . these promoters tend to have a tata box and multiple other binding sites for transcriptional regulators . nucleosome positions are more dynamic ( double - headed arrows ) and nucleosomes are not strongly excluded from any particular region , and therefore compete with transcriptional regulators at their binding sites . these promoters are thus dependent on the activity of multiple transcriptional regulators and chromatin regulators ( cr ) , which increases their mutational target size . so far we have discussed the role of promoter architecture in the sensitivity to mutations , namely whether a mutation influences gene expression and to what extent . however , expression divergence could also be directly facilitated by mechanisms that increase the mutation rate(that is , the number of mutation events per unit of time ) at particular promoters . although the determinants of local mutation rates are still poorly understood , one property that has been shown to increase mutation rates is the presence of unstable tandem repeats . a recent study revealed that about 25% of all yeast promoters contain unstable tandem repeats : short ( 1 to 150 nucleotide ) stretches of dna that are repeated head to tail . for example , tag - tag - tag - tag - tag - tag - tag is a trinucleotide repeat , with the unit tag repeated seven times . tandem repeats most often consist of short ( 2 to 6 nucleotide ) , at - rich units that are repeated 10 to 30 times , and occur frequently about 20 to 100 nucleotides upstream of the transcriptional start site . the number of repeat units changes at frequencies that are typically 10- to 10,000-fold higher than average point mutation frequencies . changes in the number of repeat units may cause gradual changes in transcription , with a certain number of units yielding maximal transcription . thus , when tandem repeats occur within promoters , their inherent instability may give rise to variants displaying altered levels of transcription , generating a pool of phenotypic diversity that allows rapid divergence . the mechanism underlying repeat - based expression divergence has been proposed to have its origins in chromatin structure . at - rich promoter repeats are known to influence local nucleosome positioning , and changes in the number of repeats affect the density and positioning of nucleosomes in the critical part of the promoter . in contrast to divergence of coding regions , divergence of gene expression can originate both from mutations in local dna sequence ( cis mutations ) - for example , a mutation that affects a promoter binding site or nucleosome position - and from mutations in other genes ( trans mutations ) , such as those encoding transcription factors or chromatin regulators . thus , increased divergence in the expression of genes could be due to their sensitivity to cis mutations or trans mutations or both . in some cases , such as variable repeat tracts , however , in other cases , the relative contribution of cis and trans mutations is unclear . for example , an increased dependence on nucleosome positioning could be due to cis mutations affecting nucleosome binding or to trans mutations affecting chromatin regulators . two approaches have been used to distinguish the effects of cis and trans mutations on gene expression on a genomic scale : genetical genomics   and analysis of hybrid species . results from both kinds of study suggest that divergence in the expression of flexible genes is due chiefly to trans mutations . for example , genes that diverged between saccharomyces cerevisiae and saccharomyces paradoxus as a result of trans mutations displayed high divergence in seven different studies comparing expression of different s. cerevisiae strains or species . in contrast , expression of genes that diverged by cis mutations displayed less divergence in the other seven studies . furthermore , the presence of a tata box or of an occupied pattern of nucleosomes ( figure 1 ) was primarily associated with increased effects of trans mutations rather than cis mutations . these results are consistent with a model in which increased flexibility of promoters is due to increased dependence on trans factors ( figure 2 ) . this could include both the number of factors that influence the expression of a given gene ( for example , a promoter occupied by nucleosomes is influenced by many chromatin regulators ) or the extent to which these factors influence expression ( tata promoters , as well as occupied promoters , could be more sensitive to the binding of transcriptional regulators ) . accordingly , promoters with particular architectures could be more tuned to the activity of various regulatory factors and thus more sensitive to evolutionary changes in their activity . notably , such promoters would also become more sensitive to variation in the activity of these regulators through physiological changes or stochastic fluctuations , which could explain the connection between expression divergence , responsiveness and noise . expression flexibility , mediated by promoter architecture , may be due to increased dependence on trans regulation and environmental changes . genes with a tata box , promoter occupied with nucleosomes and many binding sites are regulated more extensively by regulatory factors . these factors respond to extracellular signals , thus making the target genes responsive to environmental changes both on short timescales ( responsiveness and noise ) as well as on longer timescales ( evolutionary changes ) . these flexible genes preferentially code for proteins that interact with the environment and mediate the response to environmental changes ( curved arrow ) , and this may allow for rapid adaptation to new environments . expression divergence is a major driver of evolutionary change and seems to be enriched at particular genes . as described above , expression divergence in yeast correlates with several promoter features , including a large number of binding sites , a tata box , an occupied pattern of promoter nucleosomes , increased dependence on chromatin regulators and unstable tandem repeats . notably , controlling for one of these factors does not remove the effect of the others , suggesting that each of these factors have an independent effect on expression divergence . many of these factors seem to exert their influence on expression divergence predominantly through trans effects , although others ( for example , unstable repeats ) involve cis effects . as noted above , expression divergence ( the extent to which expression of a gene evolves ) correlates with expression responsiveness ( the extent to which expression of a gene is changed in response to the environment ) . we believe that the promoter elements discussed above underlie expression flexibility of these genes on short timescales ( responsiveness and noise ) , which are instrumental in the immediate response of a cell to the environment , as well as on longer timescales ( expression divergence ) , which may allow evolutionary adaptation to novel conditions . in other words , the correlation between responsiveness and expression divergence may be due to their dependence on the same promoter properties . the notion that responsive , inducible promoters differ from stable ' housekeeping ' promoters , established by struhl and colleagues [ 43,55 - 59 ] , has now been extended and linked to the evolvability of gene expression . however , for example , the protein - dna and protein - protein interactions that underlie the differential requirement of genes for general transcription factors , as well as the implications of these interactions for the dynamics of gene regulation , remain poorly understood . the fact that promoter architecture correlates with expression evolvability ( that is , the readiness with which gene expression evolves ) raises the possibility that expression evolvability may be subject to selection . this could make it possible for the expression of some genes to remain robust to mutation , whereas other genes are inherently able to change rapidly in expression under evolutionary pressure . consistent with this , we find that different promoter elements that are independently linked to expression evolvability preferentially coincide at the same genes , as if evolvability were selected in these genes . in this context , it is interesting to note that the group of rapidly diverging genes is enriched with plasma membrane genes and , in general , genes that interact with the cell environment   ( figure 2 ) . these genes are needed to cope with changes in the environment and their flexibility may allow for rapid adaptation to new environments . we apologize for omission of relevant references due to space restrictions . research in the lab of kjv is supported by the human frontier science program award hfsp rgy79/2007 , fp7 erc starting grant 241426 , vib , the ku leuven research fund and the fwo - odysseus program . research in the lab of nb is supported by the helen and martin kimmel award for innovative investigations , the eu ( funsysb ) , the israeli ministry of science and the european research council ( ideas ) .
evolutionary changes in gene expression are a main driver of phenotypic evolution . in yeast , genes that have rapidly diverged in expression 
 are associated with particular promoter features , including the presence of a tata box , a nucleosome - covered promoter and unstable tracts of tandem repeats . here 
 , we discuss how these promoter properties may confer an inherent capacity for flexibility of expression .