CiteULike: emptyhb's cis_regulatory_evolution

Genomic Inferences of the cis-Regulatory Nucleotide Polymorphisms Underlying Gene Expression Differences between Drosophila melanogaster Mating Races.

Naoki Osada — 2006-06-09T21:42:11-00:00

Mol Biol Evol (5 June 2006)

Nucleotide sequence polymorphisms affecting gene expression occur in the regulatory region of genes (in cis) and elsewhere in the genome (in trans). Further study is required to weight the relative importance of cis- and trans-acting mutations in mediating gene expression differences within and between species. Here, microarray hybridization experiments were used to isolate 363 gene expression differences between the female fly head transcriptomes of two D. melanogaster strains. One strain (French) represented the Cosmopolitan M mating race and the other strain (ZS30) represented the Z mating race derived from Zimbabwe, Africa. From chromosomal substitution strains engineered from the two strains we inferred that the expression differences between M and Z alleles largely could be attributed to the genotype of the chromosomes where the differentially expressed genes were located, i.e. cis-regulatory polymorphisms prominently influence gene expression differences between M and Z. The effects of trans-regulatory polymorphisms were apparent yet difficult to quantify. Results have implications for models of gene regulatory evolution as well as experimental studies trying to identify the nucleotide sequence polymorphisms underlying gene expression differences between Drosophila strains.

Frequent Gain and Loss of Functional Transcription Factor Binding Sites

Scott Doniger — 2007-05-26T03:59:06-00:00

PLoS Computational Biology, Vol. 3, No. 5. (1 May 2007), e99.

Cis-regulatory sequences are not always conserved across species. Divergence within cis-regulatory sequences may result from the evolution of species-specific patterns of gene expression or the flexible nature of the cis-regulatory code. The identification of functional divergence in cis-regulatory sequences is therefore important for both understanding the role of gene regulation in evolution and annotating regulatory elements. We have developed an evolutionary model to detect the loss of constraint on individual transcription factor binding sites (TFBSs). We find that a significant fraction of functionally constrained binding sites have been lost in a lineage-specific manner among three closely related yeast species. Binding site loss has previously been explained by turnover, where the concurrent gain and loss of a binding site maintains gene regulation. We estimate that nearly half of all loss events cannot be explained by binding site turnover. Recreating the mutations that led to binding site loss confirms that these sequence changes affect gene expression in some cases. We also estimate that there is a high rate of binding site gain, as more than half of experimentally identified S. cerevisiae binding sites are not conserved across species. The frequent gain and loss of TFBSs implies that cis-regulatory sequences are labile and, in the absence of turnover, may contribute to species-specific patterns of gene expression.

Functional Analysis of Repressor Binding Sites in the iab-2 Regulatory Region of the abdominal-A Homeotic Gene

Mary Shimell — 2008-07-10T15:43:08-00:00

Developmental Biology, Vol. 218, No. 1. (1 February 2000), pp. 38-52.

Spatial boundaries of homeotic gene expression are initiated and maintained by two sets of transcriptional repressors: the gap gene products and the Polycomb group proteins. Previously, the Hunchback (HB) protein has been implicated in setting the anterior expression limit of the UBX homeotic protein in parasegment 6. Here we investigate DNA elements and trans-acting repressors that control spatial expression of the Abdominal-A (ABD-A) homeotic protein. Analysis of a 1.7-kb enhancer element [iab-2(1.7)] from the iab-2 regulatory region shows that in contrast to Ubx enhancer elements, both HB and Krüppel (KR) are required to set the ABD-A anterior boundary in parasegment 7. DNase I footprinting and site-directed mutagenesis show that HB and KR are direct regulators of this iab-2 enhancer. The single KR site can be moved to a new location 100 bp away and still maintain repressive activity, whereas relocation by 300 bp abolishes activity. These results suggest that KR repression occurs through a local quenching mechanism. We also show that the gap repressor Giant (GT) initially establishes a posterior expression limit at PS9, which shifts posteriorly after the blastoderm stage. Finally, we show that this iab-2 enhancer contains multiple binding sites for the Polycomb group protein Pleiohomeotic (PHO). These iab-2 PHO sites are required in vivo for chromosome pairing-dependent repression of a mini-white reporter. However, the PHO sites are not sufficient to maintain repression of a homeotic reporter gene anterior to PS7. Full maintenance at late embryonic stages requires additional sequences adjacent to the iab-2(1.7) enhancer.

Functional Architecture and Evolution of Transcriptional Elements That Drive Gene Coexpression

Christopher Brown — 2007-09-14T17:12:44-00:00

Science, Vol. 317, No. 5844. (14 September 2007), pp. 1557-1560.

Transcriptional coexpression of interacting gene products is required for complex molecular processes; however, the function and evolution of cis-regulatory elements that orchestrate coexpression remain largely unexplored. We mutagenized 19 regulatory elements that drive coexpression of Ciona muscle genes and obtained quantitative estimates of the cis-regulatory activity of the 77 motifs that comprise these elements. We found that individual motif activity ranges broadly within and among elements, and among different instantiations of the same motif type. The activity of orthologous motifs is strongly constrained, although motif arrangement, type, and activity vary greatly among the elements of different co-regulated genes. Thus, the syntactical rules governing this regulatory function are flexible but become highly constrained evolutionarily once they are established in a particular element. 10.1126/science.1145893

Coordination of a Transcriptional Switch by HMGI(Y) Acetylation

Nikhil Munshi — 2008-07-03T22:28:56-00:00

Science, Vol. 293, No. 5532. (10 August 2001), pp. 1133-1136.

10.1126/science.293.5532.1133

An Atomic Model of the Interferon-[beta] Enhanceosome

Daniel Panne — 2008-07-03T22:25:09-00:00

Cell, Vol. 129, No. 6. (15 June 2007), pp. 1111-1123.

Summary Transcriptional activation of the interferon-[beta] (IFN-[beta]) gene requires assembly of an enhanceosome containing ATF-2/c-Jun, IRF-3/IRF-7, and NF[kappa]B. These factors bind cooperatively to the IFN-[beta] enhancer and recruit coactivators and chromatin-remodeling proteins to the IFN-[beta] promoter. We describe here a crystal structure of the DNA-binding domains of IRF-3, IRF-7, and NF[kappa]B, bound to one half of the enhancer, and use a previously described structure of the remaining half to assemble a complete picture of enhanceosome architecture in the vicinity of the DNA. Association of eight proteins with the enhancer creates a continuous surface for recognizing a composite DNA-binding element. Paucity of local protein-protein contacts suggests that cooperative occupancy of the enhancer comes from both binding-induced changes in DNA conformation and interactions with additional components such as CBP. Contacts with virtually every nucleotide pair account for the evolutionary invariance of the enhancer sequence.

Sepsid even-skipped Enhancers Are Functionally Conserved in Drosophila Despite Lack of Sequence Conservation

Emily Hare — 2008-06-27T18:46:42-00:00

PLoS Genet, Vol. 4, No. 6. (27 June 2008), e1000106.

The gene expression pattern specified by an animal regulatory sequence is generally viewed as arising from the particular arrangement of transcription factor binding sites it contains. However, we demonstrate here that regulatory sequences whose binding sites have been almost completely rearranged can still produce identical outputs. We sequenced the even-skipped locus from six species of scavenger flies (Sepsidae) that are highly diverged from the model species Drosophila melanogaster, but share its basic patterns of developmental gene expression. Although there is little sequence similarity between the sepsid eve enhancers and their well-characterized D. melanogaster counterparts, the sepsid and Drosophila enhancers drive nearly identical expression patterns in transgenic D. melanogaster embryos. We conclude that the molecular machinery that connects regulatory sequences to the transcription apparatus is more flexible than previously appreciated. In exploring this diverse collection of sequences to identify the shared features that account for their similar functions, we found a small number of short (20–30 bp) sequences nearly perfectly conserved among the species. These highly conserved sequences are strongly enriched for pairs of overlapping or adjacent binding sites. Together, these observations suggest that the local arrangement of binding sites relative to each other is more important than their overall arrangement into larger units of cis-regulatory function.

A Test of the Null Model for 5' UTR Evolution Based on GC Content

Max Reuter — 2008-04-14T19:36:59-00:00

Mol Biol Evol, Vol. 25, No. 5. (1 May 2008), pp. 801-804.

Eukaryotic mRNAs are headed by a stretch of noncoding sequence, the 5' untranslated region (UTR). It has been proposed that the length of 5' UTRs is selectively neutral and evolves under a process of stochastic destruction and recruitment of core promoter elements, combined with selection against the premature initiation of translation. We test this null model by investigating whether 5' UTR length varies with genomic GC content, an implicit prediction of the model. Using simulations, we show that the null model predicts a positive relationship between GC content and UTR length for genes regulated by a TATA box. Although this prediction is borne out qualitatively in genomic data from yeast, fruit flies, and humans, we find marked quantitative discrepancies. We conclude that UTR length may be shaped to some degree by the forces considered in the null model but that the model fails to provide a complete explanation for UTR length evolution. 10.1093/molbev/msn044

The evolution of transcription-initiation sites.

M Lynch — 2006-07-11T16:00:10-00:00

Mol Biol Evol, Vol. 22, No. 4. (April 2005), pp. 1137-1146.

Unlike the situation in prokaryotes, most eukaryotic messenger RNAs contain a moderately long 5' untranslated region (UTR). Such leader sequences impose a burden on eukaryotic genes by providing substrate for the mutational origin of premature translation-initiation codons, which generally result in defective proteins. To gain an insight into the expansion of 5' UTRs in eukaryotic genomes, we present a simple null model in which the evolution of transcription-initiation sites is entirely driven by the stochastic mutational flux of core-promoter sequences and premature translation-initiation codons. This model yields results consistent with a variety of heretofore disconnected observations, including the form of length distributions of 5' UTRs, the relatively low variance in UTR features among distantly related eukaryotes, the universal reliance on relatively simple core-promoter sequences, and the elevated density of introns in the 5' UTR. We suggest that the reduced effective population sizes of most eukaryotes impose a population-genetic environment conducive to the movement of core promoters to random positions, subject to the constraint imposed by the upstream accumulation of premature translation-initiation codons. If this hypothesis is correct, then selection for gene-specific regulatory features need not be invoked to explain either the origin of lengthy eukaryotic 5' UTRs or the 1,000-fold range of 5'-UTR lengths among genes within species. Nevertheless, once permanently established, expanded 5' UTRs may have provided a novel substrate for the evolution of mechanisms for posttranscriptional regulation of eukaryotic gene expression. These results provide a further example of how an increase in the power of random genetic drift can passively promote the evolution of forms of gene architecture that ultimately facilitate the evolution of organismal complexity.

The transcriptional consequences of mutation and natural selection in Caenorhabditis elegans

Dee Denver — 2005-04-27T18:51:48-00:00

Nature Genetics, Vol. 37, No. 5. (24 April 2005), pp. 544-548.

Evolutionary developmental biology and the problem of variation.

DL Stern — 2007-02-05T18:26:44-00:00

Evolution Int J Org Evolution, Vol. 54, No. 4. (August 2000), pp. 1079-1091.

One of the oldest problems in evolutionary biology remains largely unsolved. Which mutations generate evolutionarily relevant phenotypic variation? What kinds of molecular changes do they entail? What are the phenotypic magnitudes, frequencies of origin, and pleiotropic effects of such mutations? How is the genome constructed to allow the observed abundance of phenotypic diversity? Historically, the neo-Darwinian synthesizers stressed the predominance of micromutations in evolution, whereas others noted the similarities between some dramatic mutations and evolutionary transitions to argue for macromutationism. Arguments on both sides have been biased by misconceptions of the developmental effects of mutations. For example, the traditional view that mutations of important developmental genes always have large pleiotropic effects can now be seen to be a conclusion drawn from observations of a small class of mutations with dramatic effects. It is possible that some mutations, for example, those in cis-regulatory DNA, have few or no pleiotropic effects and may be the predominant source of morphological evolution. In contrast, mutations causing dramatic phenotypic effects, although superficially similar to hypothesized evolutionary transitions, are unlikely to fairly represent the true path of evolution. Recent developmental studies of gene function provide a new way of conceptualizing and studying variation that contrasts with the traditional genetic view that was incorporated into neo-Darwinian theory and population genetics. This new approach in developmental biology is as important for microevolutionary studies as the actual results from recent evolutionary developmental studies. In particular, this approach will assist in the task of identifying the specific mutations generating phenotypic variation and elucidating how they alter gene function. These data will provide the current missing link between molecular and phenotypic variation in natural populations.

Evolution of alternative transcriptional circuits with identical logic

Annie Tsong — 2006-09-28T08:03:10-00:00

Nature, Vol. 443, No. 7110. (2006), pp. 415-420.

Detecting the limits of regulatory element conservation and divergence estimation using pairwise and multiple alignments

Daniel Pollard — 2006-08-14T05:59:05-00:00

BMC Bioinformatics, Vol. 7 (14 August 2006), 376.

Large-scale turnover of functional transcription factor binding sites in Drosophila.

AM Moses — 2006-12-08T05:42:25-00:00

PLoS Comput Biol, Vol. 2, No. 10. (October 2006)

The gain and loss of functional transcription factor binding sites has been proposed as a major source of evolutionary change in cis-regulatory DNA and gene expression. We have developed an evolutionary model to study binding-site turnover that uses multiple sequence alignments to assess the evolutionary constraint on individual binding sites, and to map gain and loss events along a phylogenetic tree. We apply this model to study the evolutionary dynamics of binding sites of the Drosophila melanogaster transcription factor Zeste, using genome-wide in vivo (ChIP-chip) binding data to identify functional Zeste binding sites, and the genome sequences of D. melanogaster, D. simulans, D. erecta, and D. yakuba to study their evolution. We estimate that more than 5% of functional Zeste binding sites in D. melanogaster were gained along the D. melanogaster lineage or lost along one of the other lineages. We find that Zeste-bound regions have a reduced rate of binding-site loss and an increased rate of binding-site gain relative to flanking sequences. Finally, we show that binding-site gains and losses are asymmetrically distributed with respect to D. melanogaster, consistent with lineage-specific acquisition and loss of Zeste-responsive regulatory elements.

Position specific variation in the rate of evolution in transcription factor binding sites.

AM Moses — 2005-02-03T03:29:37-00:00

BMC Evol Biol, Vol. 3, No. 1. (28 August 2003)

BACKGROUND: The binding sites of sequence specific transcription factors are an important and relatively well-understood class of functional non-coding DNAs. Although a wide variety of experimental and computational methods have been developed to characterize transcription factor binding sites, they remain difficult to identify. Comparison of non-coding DNA from related species has shown considerable promise in identifying these functional non-coding sequences, even though relatively little is known about their evolution. RESULTS: Here we analyse the genome sequences of the budding yeasts Saccharomyces cerevisiae, S. bayanus, S. paradoxus and S. mikatae to study the evolution of transcription factor binding sites. As expected, we find that both experimentally characterized and computationally predicted binding sites evolve slower than surrounding sequence, consistent with the hypothesis that they are under purifying selection. We also observe position-specific variation in the rate of evolution within binding sites. We find that the position-specific rate of evolution is positively correlated with degeneracy among binding sites within S. cerevisiae. We test theoretical predictions for the rate of evolution at positions where the base frequencies deviate from background due to purifying selection and find reasonable agreement with the observed rates of evolution. Finally, we show how the evolutionary characteristics of real binding motifs can be used to distinguish them from artefacts of computational motif finding algorithms. CONCLUSION: As has been observed for protein sequences, the rate of evolution in transcription factor binding sites varies with position, suggesting that some regions are under stronger functional constraint than others. This variation likely reflects the varying importance of different positions in the formation of the protein-DNA complex. The characterization of the pattern of evolution in known binding sites will likely contribute to the effective use of comparative sequence data in the identification of transcription factor binding sites and is an important step toward understanding the evolution of functional non-coding DNA.

Sequence turnover and tandem repeats in cis-regulatory modules in drosophila.

S Sinha — 2007-10-16T22:05:08-00:00

Mol Biol Evol, Vol. 22, No. 4. (April 2005), pp. 874-885.

The path by which regulatory sequence can change, yet preserve function, is an important open question for both evolution and bioinformatics. The recent sequencing of two additional species of Drosophila plus the wealth of data on gene regulation in the fruit fly provides new means for addressing this question. For regulatory sequences, indels account for more base pairs (bp) of change than substitutions (between Drosophila melanogaster and Drosophila yakuba), though they are fewer in number. Using Drosophila pseudoobscura as an out-group, we can distinguish insertions from deletions (with maximum parsimony criteria), and find a ratio between 1 and 5 (insertions to deletions) that is species dependent and much larger than the ratio of 1/8 for neutral sequences (Petrov and Hartl 1998). Because neutral sequence is rapidly cleared from the genome, most noncoding regions which preserve their length between D. melanogaster-D. pseudoobscura and have an excess of insertions over deletions should be functional. A fraction of 15%-18% (i.e., more than 20 standard deviations from random expectation) of the regulatory sequence is covered by low copy number tandem repeats whose repeating unit has an average length of 5-10 bp and which occur preferentially (25%-45% coverage) in indels. All indels may be due to tandem repeats if we extrapolate the detection efficiency of the repeat-finding algorithms using the observed point mutation rate between the species we compare. Sequence creation by local duplication accords with the tendency for multiple copies of transcription factor-binding sites to occur in regulatory modules. Thus, indel events and tandem repeats in particular need to be incorporated into models of regulatory evolution because they can alter the rate at which beneficial variants arise and should also influence bioinformatic algorithms that parse regulatory sequences into binding sites.

Evolution of cis-regulatory sequence and function in Diptera

PJ Wittkopp — 2006-07-20T21:55:39-00:00

Heredity, Vol. aop, No. current.

The Evolution of Transcriptional Regulation in Eukaryotes

Gregory Wray — 2006-10-16T14:20:09-00:00

Mol Biol Evol, Vol. 20, No. 9. (1 September 2003), pp. 1377-1419.

Gene expression is central to the genotype-phenotype relationship in all organisms, and it is an important component of the genetic basis for evolutionary change in diverse aspects of phenotype. However, the evolution of transcriptional regulation remains understudied and poorly understood. Here we review the evolutionary dynamics of promoter, or cis-regulatory, sequences and the evolutionary mechanisms that shape them. Existing evidence indicates that populations harbor extensive genetic variation in promoter sequences, that a substantial fraction of this variation has consequences for both biochemical and organismal phenotype, and that some of this functional variation is sorted by selection. As with protein-coding sequences, rates and patterns of promoter sequence evolution differ considerably among loci and among clades for reasons that are not well understood. Studying the evolution of transcriptional regulation poses empirical and conceptual challenges beyond those typically encountered in analyses of coding sequence evolution: promoter organization is much less regular than that of coding sequences, and sequences required for the transcription of each locus reside at multiple other loci in the genome. Because of the strong context-dependence of transcriptional regulation, sequence inspection alone provides limited information about promoter function. Understanding the functional consequences of sequence differences among promoters generally requires biochemical and in vivo functional assays. Despite these challenges, important insights have already been gained into the evolution of transcriptional regulation, and the pace of discovery is accelerating. 10.1093/molbev/msg140

The evolutionary significance of cis-regulatory mutations

Gregory Wray — 2007-02-17T21:01:11-00:00

Nature Reviews Genetics, Vol. 8, No. 3., pp. 206-216.

Possibility of extensive neutral evolution under stabilizing selection with special reference to nonrandom usage of synonymous codons.

M Kimura — 2008-04-30T23:21:54-00:00

Proceedings of the National Academy of Sciences of the United States of America, Vol. 78, No. 9. (September 1981), pp. 5773-5777.

The rate of evolution in terms of the number of mutant substitutions in a finite population is investigated assuming a quantitative character subject to stabilizing selection, which is known to be the most prevalent type of natural selection. It is shown that, if a large number of segregating loci (or sites) are involved, the average selection coefficient per mutant under stabilizing selection may be exceedingly small. These mutants are very slightly deleterious but nearly neutral, so that mutant substitutions are mainly controlled by random drift, although the rate of evolution may be lower as compared with the situation in which all the mutations are strictly neutral. This is treated quantitatively by using the diffusion equation method in population genetics. A model of random drift under stabilizing selection is then applied to the problem of "nonrandom" or unequal usage of synonymous codons, and it is shown that such nonrandomness can readily be understood within the framework of the neutral mutation--random drift hypothesis (the neutral theory, for short) of molecular evolution.

Evolutionary population genetics of promoters: Predicting binding sites and functional phylogenies.

V Mustonen — 2005-11-04T19:56:37-00:00

Proc Natl Acad Sci U S A, Vol. 102, No. 44. (1 November 2005), pp. 15936-15941.

We study the evolution of transcription factor-binding sites in prokaryotes, using an empirically grounded model with point mutations and genetic drift. Selection acts on the site sequence via its binding affinity to the corresponding transcription factor. Calibrating the model with populations of functional binding sites, we verify this form of selection and show that typical sites are under substantial selection pressure for functionality: for cAMP response protein sites in Escherichia coli, the product of fitness difference and effective population size takes values 2NDeltaF of order 10. We apply this model to cross-species comparisons of binding sites in bacteria and obtain a prediction method for binding sites that uses evolutionary information in a quantitative way. At the same time, this method predicts the functional histories of orthologous sites in a phylogeny, evaluating the likelihood for conservation or loss or gain of function during evolution. We have performed, as an example, a cross-species analysis of E. coli, Salmonella typhimurium, and Yersinia pseudotuberculosis. Detailed lists of predicted sites and their functional phylogenies are available.

Evolutionary analysis of the well characterized endo16 promoter reveals substantial variation within functional sites.

JP Balhoff — 2006-12-23T17:19:35-00:00

Proc Natl Acad Sci U S A, Vol. 102, No. 24. (14 June 2005), pp. 8591-8596.

The evolutionary mechanisms that operate on genetic variation within transcriptional regulatory sequences are not well understood. We present here an evolutionary analysis of an exceptionally well characterized cis-regulatory region, the endo16 promoter of the purple sea urchin. Segregating variation reveals striking differences in the intensity of negative selection among regulatory modules, reflecting their distinct functional roles. Surprisingly, transcription-factor-binding sites are as polymorphic and as likely to contain fixed differences as flanking nucleotides. Whereas nucleotides in protein-binding sites in the most proximal regulatory module exhibit reduced variation, those in other modules tend to be more polymorphic than putatively nonfunctional nucleotides. Two unrelated large insertions at the same position within the promoter are segregating at low frequencies; one is a strong ectodermal repressor that contains 16 verified transcription-factor-binding sites. These results demonstrate that a simple relationship between conservation and function does not exist within this cis-regulatory region and highlight significant population heterogeneity in the fine structure of a well understood promoter.

Shape and function of the Bicoid morphogen gradient in dipteran species with different sized embryos.

Thomas Gregor — 2008-03-28T22:31:11-00:00

Dev Biol (13 February 2008)

The Bicoid morphogen evolved approximately 150 MYA from a Hox3 duplication and is only found in higher dipterans. A major difference between dipteran species, however, is the size of the embryo, which varies up to 5-fold. Although the expression of developmental factors scale with egg length, it remains unknown how this scaling is achieved. To test whether scaling is accounted for by the properties of Bicoid, we expressed eGFP fused to the coding region of bicoid from three dipteran species in transgenic Drosophila embryos using the Drosophila bicoid cis-regulatory and mRNA localization sequences. In such embryos, we find that Lucilia sericata and Calliphora vicina Bicoid produce gradients very similar to the endogenous Drosophila gradient and much shorter than what they would have produced in their own respective species. The common shape of the Drosophila, Lucilia and Calliphora Bicoid gradients appears to be a conserved feature of the Bicoid protein. Surprisingly, despite their similar distributions, we find that Bicoid from Lucilia and Calliphora do not rescue Drosophila bicoid mutants, suggesting that that Bicoid proteins have evolved species-specific functional amino acid differences. We also found that maternal expression and anteriorly localization of proteins other than Bcd does not necessarily give rise to a gradient; eGFP produced a uniform protein distribution. However, a shallow gradient was observed using eGFP-NLS, suggesting nuclear localization may be necessary but not sufficient for gradient formation.

Functional Evolution of a cis-Regulatory Module

Michael Ludwig — 2007-09-23T23:02:44-00:00

PLoS Biology, Vol. 3, No. 4. (1 April 2005), e93.

Lack of knowledge about how regulatory regions evolve in relation to their structure–function may limit the utility of comparative sequence analysis in deciphering cis-regulatory sequences. To address this we applied reverse genetics to carry out a functional genetic complementation analysis of a eukaryotic cis-regulatory module—the even-skipped stripe 2 enhancer—from four Drosophila species. The evolution of this enhancer is non-clock-like, with important functional differences between closely related species and functional convergence between distantly related species. Functional divergence is attributable to differences in activation levels rather than spatiotemporal control of gene expression. Our findings have implications for understanding enhancer structure–function, mechanisms of speciation and computational identification of regulatory modules.

Functional analysis of eve stripe 2 enhancer evolution in Drosophila: rules governing conservation and change.

MZ Ludwig — 2006-12-03T23:29:52-00:00

Development, Vol. 125, No. 5. (March 1998), pp. 949-958.

Experimental investigations of eukaryotic enhancers suggest that multiple binding sites and trans-acting regulatory factors are often required for wild-type enhancer function. Genetic analysis of the stripe 2 enhancer of even-skipped (eve), an important developmental gene in Drosophila, provides support for this view. Given the importance of even-skipped expression in early Drosophila development, it might be predicted that many structural features of the stripe 2 enhancer will be evolutionarily conserved, including the DNA sequences of protein binding sites and the spacing between them. To test this hypothesis, we compared sequences of the stripe 2 enhancer between four species of Drosophila: D. melanogaster, D. yakuba, D. erecta and D. pseudoobscura. Our analysis revealed a large number of nucleotide substitutions in regulatory protein binding sites for bicoid, hunchback, Kruppel and giant, as well as a systematic change in the size of the enhancer. Some of the binding sites in D. melanogaster are either absent or modified in other species. One functionally important bicoid-binding site in D. melanogaster appears to be recently evolved. We, therefore, investigated possible functional consequences of sequence differences among these stripe 2 enhancers by P-element-mediated transformation. This analysis revealed that the eve stripe 2 enhancer from each of the four species drove reporter gene expression at the identical time and location in D. melanogaster embryos. Double staining of native eve protein and transgene mRNA in early embryos showed that the reporter gene mimicked native eve expression and, in every case, produced sharply defined stripes at the blastoderm stage that were coincident with eve stripe 2 protein. We argue that stripe 2 eve expression in Drosophila evolution can be viewed as being under constant stabilizing selection with respect to the location of the anterior and posterior borders of the stripe. We further hypothesize that the stripe 2 enhancer is functionally robust, so that its evolution may be governed by the fixation of both slightly deleterious and adaptive mutations in regulatory protein binding sites as well as in the spacing between binding sites. This view allows for a slow but continual turnover of functionally important changes in the stripe 2 enhancer.

Evolutionary analysis of the cis-regulatory region of the spicule matrix gene SM50 in strongylocentrotid sea urchins.

J Walters — 2008-04-18T01:36:18-00:00

Developmental biology, Vol. 315, No. 2. (15 March 2008), pp. 567-578.

An evolutionary analysis of transcriptional regulation is essential to understanding the molecular basis of phenotypic diversity. The sea urchin is an ideal system in which to explore the functional consequence of variation in cis-regulatory sequences. We are particularly interested in the evolution of genes involved in the patterning and synthesis of its larval skeleton. This study focuses on the cis-regulatory region of SM50, which has already been characterized to a considerable extent in the purple sea urchin, Strongylocentrotus purpuratus. We have isolated the cis-regulatory region from 15 individuals of S. purpuratus as well as seven closely related species in the family Strongylocentrotidae. We have performed a variety of statistical tests and present evidence that the cis-regulatory elements upstream of the SM50 gene have been subject to positive selection along the lineage leading to S. purpuratus. In addition, we have performed electrophoretic mobility shift assays (EMSAs) and demonstrate that nucleotide substitutions within Element C affect the ability of nuclear proteins to bind to this cis-regulatory element among members of the family Strongylocentrotidae. We speculate that such changes in SM50 and other genes could accumulate to produce altered patterns of gene expression with functional consequences during skeleton formation.

A position-dependent organisation of retinoid response elements is conserved in the vertebrate Hox clusters.

G Mainguy — 2008-04-14T04:43:29-00:00

Trends in genetics : TIG, Vol. 19, No. 9. (September 2003), pp. 476-479.

Divergence of Hoxc8 early enhancer parallels diverged axial morphologies between mammals and fishes.

S Anand — 2008-04-14T04:34:44-00:00

Proceedings of the National Academy of Sciences of the United States of America, Vol. 100, No. 26. (23 December 2003), pp. 15666-15669.

There is considerable interest in understanding how cis-regulatory modifications drive morphological changes across species. Because developmental regulatory genes, including Hox genes, are remarkably conserved, their noncoding regulatory regions are likely sources for variations. Modifications of Hox cis-regulatory elements have potential to alter Hox gene expression and, hence, axial morphologies. In vertebrates, differences in the axial levels of Hox gene expression correlate with differences in the number and relative position of thoracic vertebrae. Variation in cis-regulatory elements of Hox genes can be identified by comparative sequence and reporter gene analyses in transgenic mouse embryos. Using these approaches, we show a remarkable divergence of the Hoxc8 early enhancers between mammals and fishes representing diverse axial morphologies. Extensive restructuring of the Hoxc8 early enhancer including nucleotide substitutions, inversion, and divergence result in distinct patterns of reporter gene expression along the embryonic axis. Our results provide an evolutionary perspective on how the enhancer elements are engineered and support the hypothesis that remodeling of Hox regulatory elements in different species has played a significant role in generating morphological diversity.

Comparative genomics at the vertebrate extremes.

D Boffelli — 2007-04-04T20:07:25-00:00

Nat Rev Genet, Vol. 5, No. 6. (June 2004), pp. 456-465.

The effects of selection against spurious transcription factor binding sites.

MW Hahn — 2007-10-16T21:36:07-00:00

Mol Biol Evol, Vol. 20, No. 6. (June 2003), pp. 901-906.

Most genomes contain nucleotide sequences with no known function; such sequences are assumed to be free of constraints, evolving only according to the vagaries of mutation. Here we show that selection acts to remove spurious transcription factor binding site motifs throughout 52 fully sequenced genomes of Eubacteria and Archaea. Examining the sequences necessary for polymerase binding, we find that spurious binding sites are underrepresented in both coding and noncoding regions. The average proportion of spurious binding sites found relative to the expected is 80% in eubacterial genomes and 89% in archaeal genomes. We also estimate the strength of selection against spurious binding sites in the face of the constant creation of new binding sites via mutation. Under conservative assumptions, we estimate that selection is weak, with the average efficacy of selection against spurious binding sites, Nes, of -0.12 for eubacterial genomes and -0.06 for archaeal genomes, similar to that of codon bias. Our results suggest that both coding and noncoding sequences are constrained by selection to avoid specific regions of sequence space.

Rewiring of the Yeast Transcriptional Network Through the Evolution of Motif Usage

Jan Ihmels — 2005-08-05T18:55:10-00:00

Science, Vol. 309, No. 5736. (05 August 2005), pp. 938-940.

Recent experiments revealed large-scale differences in the transcription programs of related species, yet little is known about the genetic basis underlying the evolution of gene expression and its contribution to phenotypic diversity. Here we describe a large-scale modulation of the yeast transcription program that is connected to the emergence of the capacity for rapid anaerobic growth. Genes coding for mitochondrial and cytoplasmic ribosomal proteins display a strongly correlated expression pattern in Candida albicans, but this correlation is lost in the fermentative yeast Saccharomyces cerevisiae. We provide evidence that this change in gene expression is connected to the loss of a specific cis-regulatory element from dozens of genes following the apparent whole-genome duplication event. Our results shed new light on the genetic mechanisms underlying the large-scale evolution of transcriptional networks.

Molecular mechanisms of selector gene function and evolution

Richard Mann — 2008-04-01T05:35:04-00:00

Current Opinion in Genetics & Development, Vol. 12, No. 5. (1 October 2002), pp. 592-600.

Selector proteins regulate the formation and identity of animal body regions, organs, tissues, and cell types. Recent studies have focused on the regulation of the DNA binding and transcriptional regulatory activity of this special class of transcription factors. Elucidation of the architecture of selector-regulated target gene enhancers and gene networks, and comparative studies of selector protein function are providing important insights into the evolution of development and morphology.

Evidence for stabilizing selection in a eukaryotic enhancer element

Michael Ludwig — 2006-04-22T18:30:45-00:00

Nature, Vol. 403, No. 6769. (3 February 2000), pp. 564-567.

Evidence for widespread degradation of gene control regions in hominid genomes.

PD Keightley — 2007-09-13T16:12:49-00:00

PLoS Biol, Vol. 3, No. 2. (February 2005)

Although sequences containing regulatory elements located close to protein-coding genes are often only weakly conserved during evolution, comparisons of rodent genomes have implied that these sequences are subject to some selective constraints. Evolutionary conservation is particularly apparent upstream of coding sequences and in first introns, regions that are enriched for regulatory elements. By comparing the human and chimpanzee genomes, we show here that there is almost no evidence for conservation in these regions in hominids. Furthermore, we show that gene expression is diverging more rapidly in hominids than in murids per unit of neutral sequence divergence. By combining data on polymorphism levels in human noncoding DNA and the corresponding human-chimpanzee divergence, we show that the proportion of adaptive substitutions in these regions in hominids is very low. It therefore seems likely that the lack of conservation and increased rate of gene expression divergence are caused by a reduction in the effectiveness of natural selection against deleterious mutations because of the low effective population sizes of hominids. This has resulted in the accumulation of a large number of deleterious mutations in sequences containing gene control elements and hence a widespread degradation of the genome during the evolution of humans and chimpanzees.

Evolutionary dynamics of the enhancer region of even-skipped in Drosophila.

MZ Ludwig — 2007-10-16T21:48:54-00:00

Mol Biol Evol, Vol. 12, No. 6. (November 1995), pp. 1002-1011.

We report findings on naturally occurring variation in the regulatory region of even-skipped in Drosophila. This pair-rule gene encodes a homeobox-containing transcription factor, is expressed as a series of seven transverse stripes in developing embryos, and defines parasegmental boundaries. The 5' flanking region of the gene contains a 671-bp enhancer governing stripe 2 expression. The stripe 2 enhancer contains multiple binding sites for four transcription factors that provide positional information in developing blastoderm, the positive regulators bicoid and hunchback and the repressors giant and Kruppel. The study compares polymorphism and divergence in Drosophila melanogaster and Drosophila simulans for the enhancer region, the spacer between the enhancer and the transcription start site, the untranslated leader, the first exon and the intron of eve. Contrary to expectations, we find a relatively high level of variation in the stripe 2 enhancer region, including point substitutions and insertion/deletions in binding sites, and a comparable level of variation in the other noncoding regions. The patterns of variation and divergence within the enhancer region and between regions of the locus fit a model of neutral molecular evolution. We suggest that the multiplicity of binding sites in the enhancer provides a redundancy in function that allows flexibility in the sequence requirements and structural design of the promoter.

Tracing the evolutionary history of Drosophila regulatory regions with models that identify transcription factor binding sites.

ET Dermitzakis — 2007-10-16T21:12:19-00:00

Mol Biol Evol, Vol. 20, No. 5. (May 2003), pp. 703-714.

Much of evolutionary change is mediated at the level of gene expression, yet our understanding of regulatory evolution remains unsatisfying. In light of recent data indicating that transcription factor binding sites undergo substantial turnover between species, we attempt to quantify the process of binding site turnover in regulatory regions of well-studied genes controlling embryonic patterning in Drosophila. We examine polymorphism and divergence data in Drosophila melanogaster and four related species from regulatory regions of five early development genes for which functional binding sites have been identified. This analysis reveals that Drosophila regulatory regions exhibit patterns of variation consistent with functional constraint. We develop a novel approach to binding site prediction which we use to characterize the process of binding site divergence in regulatory regions. This method uses sets of known binding sites to construct a model that predicts transcription factor specificity and bootstrap sampling to derive significance levels. This approach allows appropriate significance levels to be determined even in the face of skewed base composition in the background sequence. Using this approach, we show that, although functional elements exhibit conservation of sequence, there is substantial potential to gain new functional elements within the regulatory regions. Our results show that application of models that predict transcription factor binding sites can yield insights into the process and dynamics of binding site evolution within regulatory regions.

Colloquium Papers: Emerging principles of regulatory evolution.

B Prud'homme — 2007-06-14T22:46:27-00:00

Proc Natl Acad Sci U S A, Vol. 104 Suppl 1 (15 May 2007), pp. 8605-8612.

Understanding the genetic and molecular mechanisms governing the evolution of morphology is a major challenge in biology. Because most animals share a conserved repertoire of body-building and -patterning genes, morphological diversity appears to evolve primarily through changes in the deployment of these genes during development. The complex expression patterns of developmentally regulated genes are typically controlled by numerous independent cis-regulatory elements (CREs). It has been proposed that morphological evolution relies predominantly on changes in the architecture of gene regulatory networks and in particular on functional changes within CREs. Here, we discuss recent experimental studies that support this hypothesis and reveal some unanticipated features of how regulatory evolution occurs. From this growing body of evidence, we identify three key operating principles underlying regulatory evolution, that is, how regulatory evolution: (i) uses available genetic components in the form of preexisting and active transcription factors and CREs to generate novelty; (ii) minimizes the penalty to overall fitness by introducing discrete changes in gene expression; and (iii) allows interactions to arise among any transcription factor and downstream CRE. These principles endow regulatory evolution with a vast creative potential that accounts for both relatively modest morphological differences among closely related species and more profound anatomical divergences among groups at higher taxonomical levels.

Divergence of Transcription Factor Binding Sites Across Related Yeast Species

Anthony Borneman — 2007-08-10T08:47:50-00:00

Science, Vol. 317, No. 5839. (10 August 2007), pp. 815-819.

Characterization of interspecies differences in gene regulation is crucial for understanding the molecular basis of both phenotypic diversity and evolution. By means of chromatin immunoprecipitation and DNA microarray analysis, the divergence in the binding sites of the pseudohyphal regulators Ste12 and Tec1 was determined in the yeasts Saccharomyces cerevisiae, S. mikatae, and S. bayanus under pseudohyphal conditions. We have shown that most of these sites have diverged across these species, far exceeding the interspecies variation in orthologous genes. A group of Ste12 targets was shown to be bound only in S. mikatae and S. bayanus under pseudohyphal conditions. Many of these genes are targets of Ste12 during mating in S. cerevisiae, indicating that specialization between the two pathways has occurred in this species. Transcription factor binding sites have therefore diverged substantially faster than ortholog content. Thus, gene regulation resulting from transcription factor binding is likely to be a major cause of divergence between related species. 10.1126/science.1140748

Two or Four Bristles: Functional Evolution of an Enhancer of scute in Drosophilidae

Sylvain Marcellini — 2006-11-19T04:55:58-00:00

PLoS Biology, Vol. 4, No. 12. (1 November 2006), e386.

Changes in cis-regulatory sequences are proposed to underlie much of morphological evolution. Yet, little is known about how such modifications translate into phenotypic differences. To address this problem, we focus on the dorsocentral bristles of Drosophilidae. In Drosophila melanogaster, development of these bristles depends on a cis-regulatory element, the dorsocentral enhancer, to activate scute in a cluster of cells from which two bristles on the posterior scutum arise. A few species however, such as D. quadrilineata, bear anterior dorsocentral bristles as well as posterior ones, a derived feature. This correlates with an anterior expansion of the scute expression domain. Here, we show that the D. quadrilineata enhancer has evolved, and is now active in more anterior regions. When used to rescue scute expression in transgenic D. melanogaster, the D. quadrilineata enhancer is able to induce anterior bristles. Importantly, these properties are not displayed by homologous enhancers from control species bearing only two posterior bristles. We also provide evidence that upstream regulation of the enhancer, by the GATA transcription factor Pannier, has been evolutionarily conserved. This work illustrates how, in the context of a conserved trans-regulatory landscape, evolutionary tinkering of pre-existing enhancers can modify gene expression patterns and contribute to morphological diversification.

Subfunctionalization of Duplicated Zebrafish pax6 Genes by cis-Regulatory Divergence

Dirk Kleinjan — 2008-02-24T04:18:14-00:00

PLoS Genetics, Vol. 4, No. 2. (1 February 2008), e29.

Gene duplication is a major driver of evolutionary divergence. In most vertebrates a single PAX6 gene encodes a transcription factor required for eye, brain, olfactory system, and pancreas development. In zebrafish, following a postulated whole-genome duplication event in an ancestral teleost, duplicates pax6a and pax6b jointly fulfill these roles. Mapping of the homozygously viable eye mutant sunrise identified a homeodomain missense change in pax6b, leading to loss of target binding. The mild phenotype emphasizes role-sharing between the co-orthologues. Meticulous mapping of isolated BACs identified perturbed synteny relationships around the duplicates. This highlights the functional conservation of pax6 downstream (3′) control sequences, which in most vertebrates reside within the introns of a ubiquitously expressed neighbour gene, ELP4, whose pax6a-linked exons have been lost in zebrafish. Reporter transgenic studies in both mouse and zebrafish, combined with analysis of vertebrate sequence conservation, reveal loss and retention of specific cis-regulatory elements, correlating strongly with the diverged expression of co-orthologues, and providing clear evidence for evolution by subfunctionalization.

An evolutionary constraint: Strongly disfavored class of change in DNA sequence during divergence of cis-regulatory modules

Andrew Cameron — 2007-10-15T11:53:00-00:00

Proceedings of the National Academy of Sciences, Vol. 102, No. 33. (16 August 2005), pp. 11769-11774.

The DNA of functional cis-regulatory modules displays extensive sequence conservation in comparisons of genomes from modestly distant species. Patches of sequence that are several hundred base pairs in length within these modules are often seen to be 80-95% identical, although the flanking sequence cannot even be aligned. However, it is unlikely that base pairs located between the transcription factor target sites of cis-regulatory modules have sequence-dependent function, and the mechanism that constrains evolutionary change within cis-regulatory modules is incompletely understood. We chose five functionally characterized cis-regulatory modules from the Strongylocentrotus purpuratus (sea urchin) genome and obtained orthologous regulatory and flanking sequences from a bacterial artificial chromosome genome library of a congener, Strongylocentrotus franciscanus. As expected, single-nucleotide substitutions and small indels occur freely at many positions within the regulatory modules of these two species, as they do outside the regulatory modules. However, large indels (>20 bp) are statistically almost absent within the regulatory modules, although they are common in flanking intergenic or intronic sequence. The result helps to explain the patterns of evolutionary sequence divergence characteristic of cis-regulatory DNA. 10.1073/pnas.0505291102

Chance caught on the wing: cis-regulatory evolution and the origin of pigment patterns in Drosophila.

N Gompel — 2006-01-18T01:41:02-00:00

Nature, Vol. 433, No. 7025. (3 February 2005), pp. 481-487.

The gain, loss or modification of morphological traits is generally associated with changes in gene regulation during development. However, the molecular bases underlying these evolutionary changes have remained elusive. Here we identify one of the molecular mechanisms that contributes to the evolutionary gain of a male-specific wing pigmentation spot in Drosophila biarmipes, a species closely related to Drosophila melanogaster. We show that the evolution of this spot involved modifications of an ancestral cis-regulatory element of the yellow pigmentation gene. This element has gained multiple binding sites for transcription factors that are deeply conserved components of the regulatory landscape controlling wing development, including the selector protein Engrailed. The evolutionary stability of components of regulatory landscapes, which can be co-opted by chance mutations in cis-regulatory elements, might explain the repeated evolution of similar morphological patterns, such as wing pigmentation patterns in flies.

Close sequence comparisons are sufficient to identify human cis-regulatory elements.

Shyam Prabhakar — 2006-06-16T19:46:25-00:00

Genome Res (12 June 2006)

Cross-species DNA sequence comparison is the primary method used to identify functional noncoding elements in human and other large genomes. However, little is known about the relative merits of evolutionarily close and distant sequence comparisons. To address this problem, we identified evolutionarily conserved noncoding regions in primate, mammalian, and more distant comparisons using a uniform approach (Gumby) that facilitates unbiased assessment of the impact of evolutionary distance on predictive power. We benchmarked computational predictions against previously identified cis-regulatory elements at diverse genomic loci and also tested numerous extremely conserved human-rodent sequences for transcriptional enhancer activity using an in vivo enhancer assay in transgenic mice. Human regulatory elements were identified with acceptable sensitivity (53%-80%) and true-positive rate (27%-67%) by comparison with one to five other eutherian mammals or six other simian primates. More distant comparisons (marsupial, avian, amphibian, and fish) failed to identify many of the empirically defined functional noncoding elements. Our results highlight the practical utility of close sequence comparisons, and the loss of sensitivity entailed by more distant comparisons. We derived an intuitive relationship between ancient and recent noncoding sequence conservation from whole-genome comparative analysis that explains most of the observations from empirical benchmarking. Lastly, we determined that, in addition to strength of conservation, genomic location and/or density of surrounding conserved elements must also be considered in selecting candidate enhancers for in vivo testing at embryonic time points.

Evolution of genes and genomes on the Drosophila phylogeny

2007-11-07T19:25:43-00:00

Nature, Vol. 450, No. 7167. (November 2007), pp. 203-218.

Conservation of regulatory elements between two species of Drosophila.

E Emberly — 2006-09-28T20:46:21-00:00

BMC Bioinformatics, Vol. 4 (20 November 2003)

BACKGROUND: One of the important goals in the post-genomic era is to determine the regulatory elements within the non-coding DNA of a given organism's genome. The identification of functional cis-regulatory modules has proven difficult since the component factor binding sites are small and the rules governing their arrangement are poorly understood. However, the genomes of suitably diverged species help to predict regulatory elements based on the generally accepted assumption that conserved blocks of genomic sequence are likely to be functional. To judge the efficacy of strategies that prefilter by sequence conservation it is important to know to what extent the converse assumption holds, namely that functional elements common to both species will fall within these conserved blocks. The recently completed sequence of a second Drosophila species provides an opportunity to test this assumption for one of the experimentally best studied regulatory networks in multicellular organisms, the body patterning of the fly embryo. RESULTS: We find that 50%-70% of known binding sites reside in conserved sequence blocks, but these percentages are not greatly enriched over what is expected by chance. Finally, a computational genome-wide search in both species for regulatory modules based on clusters of binding sites suggests that genes central to the regulatory network are consistently recovered. CONCLUSIONS: Our results indicate that binding sites remain clustered for these "core modules" while not necessarily residing in conserved blocks. This is an important clue as to how regulatory information is encoded in the genome and how modules evolve.

Morphological evolution through multiple cis-regulatory mutations at a single gene

Alistair Mcgregor — 2007-07-17T11:11:14-00:00

Nature (15 July 2007)

Evolution at two levels in humans and chimpanzees.

MC King — 2006-03-21T22:31:51-00:00

Science, Vol. 188, No. 4184. (11 April 1975), pp. 107-116.

Repeated morphological evolution through cis-regulatory changes in a pleiotropic gene

Benjamin Prud'homme — 2006-04-20T00:45:36-00:00

Nature, Vol. 440, No. 7087., pp. 1050-1053.

Animal MicroRNAs Confer Robustness to Gene Expression and Have a Significant Impact on 3'UTR Evolution.

Alexander Stark — 2005-12-14T13:18:01-00:00

Cell (7 December 2005)

MicroRNAs are small noncoding RNAs that serve as posttranscriptional regulators of gene expression in higher eukaryotes. Their widespread and important role in animals is highlighted by recent estimates that 20%-30% of all genes are microRNA targets. Here, we report that a large set of genes involved in basic cellular processes avoid microRNA regulation due to short 3'UTRs that are specifically depleted of microRNA binding sites. For individual microRNAs, we find that coexpressed genes avoid microRNA sites, whereas target genes and microRNAs are preferentially expressed in neighboring tissues. This mutually exclusive expression argues that microRNAs confer accuracy to developmental gene-expression programs, thus ensuring tissue identity and supporting cell-lineage decisions.

Evolution of developmental genes: molecular microevolution of enhancer sequences at the Ubx locus in Drosophila and its impact on developmental phenotypes.

J Phinchongsakuldit — 2006-12-23T17:31:53-00:00

Mol Biol Evol, Vol. 21, No. 2. (February 2004), pp. 348-363.

Homeotic genes, which function to specify segment identity along the anterior-posterior axis of embryos, are controlled by extensive batteries of enhancer sequences. We have investigated patterns of interspecific and intraspecific molecular variation in three enhancers of the Ultrabithorax (Ubx) locus, which are bx-32.8, pbx32.7, and bxd4.1, from the Drosophila melanogaster species group. These enhancer sequences control Ubx expression by binding to multiple transcription factors encoded by gap, pair-rule, and dorsoventrally expressed genes. Sequence comparisons reveal purifying selection acting on all three enhancers, both in bases binding transcription factors and in bases whose functions are as yet unknown. Neutrality tests largely fail to reject a neutral evolution model. However, using a matrix similarity value to reflect the binding affinity of the protein-binding sites, interspecific and intraspecific variation that may have potential to affect the binding affinity of the sequences homologous to those binding transcription factors in D. melanogaster are discovered, suggesting evolutionary flexibility in the way in which these sequences function in the control of development. As a means of measuring the impact of intraspecific variation on observable phenotypes, we have induced Ubx mutant phenocopies with embryonic ether treatment, and find strong and highly significant variation between D. melanogaster strains in their phenocopy frequencies. This variation shows no significant correlation with the strengths of the mutant phenotypes when the strains are heterozygous with a Ubx null mutation. Estimated phylogenetic trees have been constructed for the three enhancer regions investigated. Neither of the two phenotypic traits investigated shows any significant associations with the phylogeny of any of the three enhancers.