Variation In Regulatory Sequences Research Articles

Hybrid allele-specific ChIP-seq analysis identifies variation in brassinosteroid-responsive transcription factor binding linked to traits in maize

BackgroundGenetic variation in regulatory sequences that alter transcription factor (TF) binding is a major cause of phenotypic diversity. Brassinosteroid is a growth hormone that has major effects on plant phenotypes. Genetic variation in brassinosteroid-responsive cis-elements likely contributes to trait variation. Pinpointing such regulatory variations and quantitative genomic analysis of the variation in TF-target binding, however, remains challenging. How variation in transcriptional targets of signaling pathways such as the brassinosteroid pathway contributes to phenotypic variation is an important question to be investigated with innovative approaches.ResultsHere, we use a hybrid allele-specific chromatin binding sequencing (HASCh-seq) approach and identify variations in target binding of the brassinosteroid-responsive TF ZmBZR1 in maize. HASCh-seq in the B73xMo17 F1s identifies thousands of target genes of ZmBZR1. Allele-specific ZmBZR1 binding (ASB) has been observed for 18.3% of target genes and is enriched in promoter and enhancer regions. About a quarter of the ASB sites correlate with sequence variation in BZR1-binding motifs and another quarter correlate with haplotype-specific DNA methylation, suggesting that both genetic and epigenetic variations contribute to the high level of variation in ZmBZR1 occupancy. Comparison with GWAS data shows linkage of hundreds of ASB loci to important yield and disease-related traits.ConclusionOur study provides a robust method for analyzing genome-wide variations of TF occupancy and identifies genetic and epigenetic variations of the brassinosteroid response transcription network in maize.

The evolution, evolvability and engineering of gene regulatory DNA.

Mutations in non-coding regulatory DNA sequences can alter gene expression, organismal phenotype and fitness1-3. Constructing complete fitness landscapes, in which DNA sequences are mapped to fitness, is a long-standing goal in biology, but has remained elusive because it is challenging to generalize reliably to vast sequence spaces4-6. Here we build sequence-to-expression models that capture fitness landscapes and use them to decipher principles of regulatory evolution. Using millions of randomly sampled promoter DNA sequences and their measured expression levels in the yeast Saccharomyces cerevisiae, we learn deep neural network models that generalize with excellent prediction performance, and enable sequence design for expression engineering. Using our models, we study expression divergence under genetic drift and strong-selection weak-mutation regimes to find that regulatory evolution is rapid and subject to diminishing returns epistasis; that conflicting expression objectives in different environments constrain expression adaptation; and that stabilizing selection on gene expression leads to the moderation of regulatory complexity. We present an approach for using suchmodels to detect signatures of selection on expression from natural variation in regulatory sequences and use it to discover an instance of convergent regulatory evolution. We assess mutational robustness, finding that regulatory mutation effect sizes follow a power law, characterize regulatory evolvability, visualize promoter fitness landscapes, discover evolvability archetypes and illustrate the mutational robustness of natural regulatory sequence populations. Our work provides a general framework for designing regulatorysequences andaddressing fundamental questions in regulatory evolution.

Comprehensive whole genome sequence analyses yields novel genetic and structural insights for Intellectual Disability

BackgroundIntellectual Disability (ID) is among the most common global disorders, yet etiology is unknown in ~30% of patients despite clinical assessment. Whole genome sequencing (WGS) is able to interrogate the entire genome, providing potential to diagnose idiopathic patients.MethodsWe conducted WGS on eight children with idiopathic ID and brain structural defects, and their normal parents; carrying out an extensive data analyses, using standard and discovery approaches.ResultsWe verified de novo pathogenic single nucleotide variants (SNV) in ARID1B c.1595delG and PHF6 c.820C > T, potentially causative de novo two base indels in SQSTM1 c.115_116delinsTA and UPF1 c.1576_1577delinsA, and de novo SNVs in CACNB3 c.1289G > A, and SPRY4 c.508 T > A, of uncertain significance. We report results from a large secondary control study of 2081 exomes probing the pathogenicity of the above genes. We analyzed structural variation by four different algorithms including de novo genome assembly. We confirmed a likely contributory 165 kb de novo heterozygous 1q43 microdeletion missed by clinical microarray. The de novo assembly resulted in unmasking hidden genome instability that was missed by standard re-alignment based algorithms. We also interrogated regulatory sequence variation for known and hypothesized ID genes and present useful strategies for WGS data analyses for non-coding variation.ConclusionThis study provides an extensive analysis of WGS in the context of ID, providing genetic and structural insights into ID and yielding diagnoses.

Bayesian Inference of Allele-Specific Gene Expression Indicates Abundant Cis-Regulatory Variation in Natural Flycatcher Populations.

Polymorphism in cis-regulatory sequences can lead to different levels of expression for the two alleles of a gene, providing a starting point for the evolution of gene expression. Little is known about the genome-wide abundance of genetic variation in gene regulation in natural populations but analysis of allele-specific expression (ASE) provides a means for investigating such variation. We performed RNA-seq of multiple tissues from population samples of two closely related flycatcher species and developed a Bayesian algorithm that maximizes data usage by borrowing information from the whole data set and combines several SNPs per transcript to detect ASE. Of 2,576 transcripts analyzed in collared flycatcher, ASE was detected in 185 (7.2%) and a similar frequency was seen in the pied flycatcher. Transcripts with statistically significant ASE commonly showed the major allele in >90% of the reads, reflecting that power was highest when expression was heavily biased toward one of the alleles. This would suggest that the observed frequencies of ASE likely are underestimates. The proportion of ASE transcripts varied among tissues, being lowest in testis and highest in muscle. Individuals often showed ASE of particular transcripts in more than one tissue (73.4%), consistent with a genetic basis for regulation of gene expression. The results suggest that genetic variation in regulatory sequences commonly affects gene expression in natural populations and that it provides a seedbed for phenotypic evolution via divergence in gene expression.

Plasticity and innovation of regulatory mechanisms underlying seed oil content mediated by duplicated genes in the palaeopolyploid soybean.

Many plants have undergone whole genome duplication (WGD). However, how regulatory networks underlying a particular trait are reshaped in polyploids has not been experimentally investigated. Here we show that the regulatory pathways modulating seed oil content, which involve WRINKLED1 (WRI1), LEAFY COTYLEDON1 (LEC1), and LEC2 in Arabidopsis, have been modified in the palaeopolyploid soybean. Such modifications include functional reduction of GmWRI1b of the GmWRI1a/GmWRI1b homoeologous pair relevant to WRI1, complementary non-allelic dosage effects of the GmLEC1a/GmLEC1b homoeologous pair relevant to LEC1, pseudogenization of the singleton GmLEC2 relevant to LEC2, and the rise of the LEC2-like function of GmABI3b, contrasting to its homoeolog GmABI3a, which maintains the ABSCISIC ACID INSENSITIVE 3 (ABI3)-like function in modulating seed maturation and dormancy. The function of GmABI3b in modulating seed oil biosynthesis was fulfilled by direct binding to a RY (CATGCA) cis-regulatory element in the GmWRI1a promoter, which was absent in the GmWRI1b promoter, resulting in reduction of the GmWRI1b expression. Nevertheless, the three regulators each exhibited similar intensities of purifying selection to their respective duplicates since these pairs were formed by a WGD event that is proposed to have occurred approximately 13 million years ago (mya), suggesting that the differentiation in spatiotemporal expression between the duplicated genes is more likely to be the outcome of neutral variation in regulatory sequences. This study thus exemplifies the plasticity, dynamics, and novelty of regulatory networks mediated by WGD.

2015 Curt Stern Award Introduction: Leonid Kruglyak

I am pleased to introduce Leonid Kruglyak as this year’s winner of the Curt Stern Award. The Curt Stern Award honors the memory of an outstanding and pioneering human geneticist and is presented yearly for outstanding scientific achievements that occurred in human genetics during a 10 year period in a researcher’s career. Leonid began his career in science not as a geneticist, but rather as a physicist. He graduated with highest honors in physics from Princeton University in 1987. He then got his Ph.D. in biophysics from the University of California, Berkeley, in 1990 and studied theoretical physics at NATO. He followed that with a stint as a Whitehead fellow, when he finally saw the light and joined us in the world of genetics. Leonid has held faculty positions at the Fred Hutchinson Cancer Research Center, Princeton University, and the University of California, Los Angeles, where he remains today. He is also a Howard Hughes Investigator. Needless to say, Leonid has won an extraordinary number of award and honors that cover pages of his CV. But rather than talk about all that, I’d rather tell you about his science. Leonid’s Kruglyak’s original and creative work has ranged broadly over linkage genetics, population genetics, and genomics and has drawn on experimental, mathematical, and computational approaches. The results in each area have had an extremely wide impact. His 1995 paper describing statistical standards for genome-wide linkage studies has been cited over 3,700 times and is the gold standard for interpreting putative evidence of linkage. His 1996 paper describing the program GENEHUNTER has been cited over 2,300 times. This paper was particularly important because it gave us a way to analyze human pedigrees for disease loci when no model was apparent—thus becoming the standard for nonparametric genetic mapping across human and model-organism fields. His 1999 paper on the number of SNPs needed for genome-wide association studies originally proved very controversial, but it has now been proved many times over and has laid the foundation for the HapMap project. Finally, his 2002 paper reporting the first experiment on genetics of global gene expression, and the papers that grew out of that work, have provided key insights into the genetic basis of complex traits, regulatory sequence variation, gene regulation, and the evolution of expression differences. For these and so many more reasons, he is this year’s winner of the Curt Stern Award.

Genome-wide association analysis on normal hearing function identifies PCDH20 and SLC28A3 as candidates for hearing function and loss.

Hearing loss and individual differences in normal hearing both have a substantial genetic basis. Although many new genes contributing to deafness have been identified, very little is known about genes/variants modulating the normal range of hearing ability. To fill this gap, we performed a two-stage meta-analysis on hearing thresholds (tested at 0.25, 0.5, 1, 2, 4, 8 kHz) and on pure-tone averages (low-, medium- and high-frequency thresholds grouped) in several isolated populations from Italy and Central Asia (total N = 2636). Here, we detected two genome-wide significant loci close to PCDH20 and SLC28A3 (top hits: rs78043697, P = 4.71E−10 and rs7032430, P = 2.39E−09, respectively). For both loci, we sought replication in two independent cohorts: B58C from the UK (N = 5892) and FITSA from Finland (N = 270). Both loci were successfully replicated at a nominal level of significance (P < 0.05). In order to confirm our quantitative findings, we carried out RT-PCR and reported RNA-Seq data, which showed that both genes are expressed in mouse inner ear, especially in hair cells, further suggesting them as good candidates for modulatory genes in the auditory system. Sequencing data revealed no functional variants in the coding region of PCDH20 or SLC28A3, suggesting that variation in regulatory sequences may affect expression. Overall, these results contribute to a better understanding of the complex mechanisms underlying human hearing function.

Is variation in copy number of the human beta defensin gene cluster associated with preterm birth?

BackgroundIncreased expression of antimicrobial peptides including human beta defensins (HBD) has been reported in the amniotic fluid and vaginal secretions of women who deliver preterm. We have previously shown that these women have increased first trimester serum HBD2. The gene encoding HBD2, DEFB4A, is part of a defensin beta (DEFB) cluster on chromosome 8 that is variable in copy number. Increased serum HBD2 is associated with increased DEFB copy number. We aimed to test the hypothesis that variation in DEFB copy number is associated with preterm birth. MethodsIn a retrospective, case-control study, genomic DNA and serum were extracted from blood collected from white European women at 11–13 weeks' gestation attending King's College Hospital between March 1, 2006, and Sept 30, 2010. DEFB copy number was determined by paralogue ratio test. Serum HBD2 concentration was measured by ELISA. Data were analysed with Pearson correlation (Excel, version 2010) and binary logistic regression (SPSS, version 20). FindingsCases were 102 women who either delivered preterm in the index pregnancy or had a history of preterm delivery. Controls were 152 women who had had at least one previous term delivery and delivered at term in the index pregnancy; they had no history of preterm birth. Modal copy number was 4 (range 2–7). Serum was available from 140 women (30 cases, 54 controls, 56 not included in the genetic association study). Median HBD2 concentration was 761·5 pg/mL (IQR 449·6–1232·0). There was no association between DEFB copy number and preterm birth, nor was there a correlation between copy number and serum HBD2 concentration. InterpretationAlthough variation in HBD2 protein expression in the first trimester might be useful to predict risk of preterm birth, we found no association between DEFB copy number and preterm birth. Nor did we find a correlation between DEFB copy number and serum HBD2 expression in the first trimester of pregnancy; this might be due to variation in regulatory sequences—some of which are progesterone and oestrogen sensitive—between individual copies. FundingWellcome Trust, Wellbeing of Women.

Extensive divergence of transcription factor binding in Drosophila embryos with highly conserved gene expression.

To better characterize how variation in regulatory sequences drives divergence in gene expression, we undertook a systematic study of transcription factor binding and gene expression in blastoderm embryos of four species, which sample much of the diversity in the 40 million-year old genus Drosophila: D. melanogaster, D. yakuba, D. pseudoobscura and D. virilis. We compared gene expression, measured by mRNA-seq, to the genome-wide binding, measured by ChIP-seq, of four transcription factors involved in early anterior-posterior patterning. We found that mRNA levels are much better conserved than individual transcription factor binding events, and that changes in a gene's expression were poorly explained by changes in adjacent transcription factor binding. However, highly bound sites, sites in regions bound by multiple factors and sites near genes are conserved more frequently than other binding, suggesting that a considerable amount of transcription factor binding is weakly or non-functional and not subject to purifying selection.

Maps of open chromatin highlight cell type–restricted patterns of regulatory sequence variation at hematological trait loci

Nearly three-quarters of the 143 genetic signals associated with platelet and erythrocyte phenotypes identified by meta-analyses of genome-wide association (GWA) studies are located at non-protein-coding regions. Here, we assessed the role of candidate regulatory variants associated with cell type–restricted, closely related hematological quantitative traits in biologically relevant hematopoietic cell types. We used formaldehyde-assisted isolation of regulatory elements followed by next-generation sequencing (FAIRE-seq) to map regions of open chromatin in three primary human blood cells of the myeloid lineage. In the precursors of platelets and erythrocytes, as well as in monocytes, we found that open chromatin signatures reflect the corresponding hematopoietic lineages of the studied cell types and associate with the cell type–specific gene expression patterns. Dependent on their signal strength, open chromatin regions showed correlation with promoter and enhancer histone marks, distance to the transcription start site, and ontology classes of nearby genes. Cell type–restricted regions of open chromatin were enriched in sequence variants associated with hematological indices. The majority (63.6%) of such candidate functional variants at platelet quantitative trait loci (QTLs) coincided with binding sites of five transcription factors key in regulating megakaryopoiesis. We experimentally tested 13 candidate regulatory variants at 10 platelet QTLs and found that 10 (76.9%) affected protein binding, suggesting that this is a frequent mechanism by which regulatory variants influence quantitative trait levels. Our findings demonstrate that combining large-scale GWA data with open chromatin profiles of relevant cell types can be a powerful means of dissecting the genetic architecture of closely related quantitative traits.

Copy Number Variants in Candidate Genes Are Genetic Modifiers of Hirschsprung Disease

Hirschsprung disease (HSCR) is a neurocristopathy characterized by absence of intramural ganglion cells along variable lengths of the gastrointestinal tract. The HSCR phenotype is highly variable with respect to gender, length of aganglionosis, familiality and the presence of additional anomalies. By molecular genetic analysis, a minimum of 11 neuro-developmental genes (RET, GDNF, NRTN, SOX10, EDNRB, EDN3, ECE1, ZFHX1B, PHOX2B, KIAA1279, TCF4) are known to harbor rare, high-penetrance mutations that confer a large risk to the bearer. In addition, two other genes (RET, NRG1) harbor common, low-penetrance polymorphisms that contribute only partially to risk and can act as genetic modifiers. To broaden this search, we examined whether a set of 67 proven and candidate HSCR genes harbored additional modifier alleles. In this pilot study, we utilized a custom-designed array CGH with ∼33,000 test probes at an average resolution of ∼185 bp to detect gene-sized or smaller copy number variants (CNVs) within these 67 genes in 18 heterogeneous HSCR patients. Using stringent criteria, we identified CNVs at three loci (MAPK10, ZFHX1B, SOX2) that are novel, involve regulatory and coding sequences of neuro-developmental genes, and show association with HSCR in combination with other congenital anomalies. Additional CNVs are observed under relaxed criteria. Our research suggests a role for CNVs in HSCR and, importantly, emphasizes the role of variation in regulatory sequences. A much larger study will be necessary both for replication and for identifying the full spectrum of small CNV effects.

Research Highlights

Research Highlights

Comprehensive analysis of Arabidopsis expression level polymorphisms with simple inheritance

In Arabidopsis thaliana, gene expression level polymorphisms (ELPs) between natural accessions that exhibit simple, single locus inheritance are promising quantitative trait locus (QTL) candidates to explain phenotypic variability. It is assumed that such ELPs overwhelmingly represent regulatory element polymorphisms. However, comprehensive genome-wide analyses linking expression level, regulatory sequence and gene structure variation are missing, preventing definite verification of this assumption. Here, we analyzed ELPs observed between the Eil-0 and Lc-0 accessions. Compared with non-variable controls, 5′ regulatory sequence variation in the corresponding genes is indeed increased. However, ∼42% of all the ELP genes also carry major transcription unit deletions in one parent as revealed by genome tiling arrays, representing a >4-fold enrichment over controls. Within the subset of ELPs with simple inheritance, this proportion is even higher and deletions are generally more severe. Similar results were obtained from analyses of the Bay-0 and Sha accessions, using alternative technical approaches. Collectively, our results suggest that drastic structural changes are a major cause for ELPs with simple inheritance, corroborating experimentally observed indel preponderance in cloned Arabidopsis QTL.

Regulatory Polymorphisms and their Contribution to Interindividual Differences in the Expression of Enzymes Influencing Drug and Toxicant Disposition

The unexpected paucity of human protein encoding genes suggested that polymorphisms altering gene expression might be more important than initially thought. From an evolutionary perspective, traits such as xenobiotic metabolism and transport that require a dynamic response to environmental changes would evolve more efficiently through variation in regulatory sequences versus coding variants. Such variation would be manifest as co-dominant traits and selection pressures would operate more efficiently because of their ability to impact fitness in the heterozygous state. Our current understanding of regulatory polymorphisms impacting drug disposition is reviewed including specific discussion regarding knowledge gaps and future research opportunities.

In Silico Detection of Sequence Variations Modifying Transcriptional Regulation

Identification of functional genetic variation associated with increased susceptibility to complex diseases can elucidate genes and underlying biochemical mechanisms linked to disease onset and progression. For genes linked to genetic diseases, most identified causal mutations alter an encoded protein sequence. Technological advances for measuring RNA abundance suggest that a significant number of undiscovered causal mutations may alter the regulation of gene transcription. However, it remains a challenge to separate causal genetic variations from linked neutral variations. Here we present an in silico driven approach to identify possible genetic variation in regulatory sequences. The approach combines phylogenetic footprinting and transcription factor binding site prediction to identify variation in candidate cis-regulatory elements. The bioinformatics approach has been tested on a set of SNPs that are reported to have a regulatory function, as well as background SNPs. In the absence of additional information about an analyzed gene, the poor specificity of binding site prediction is prohibitive to its application. However, when additional data is available that can give guidance on which transcription factor is involved in the regulation of the gene, the in silico binding site prediction improves the selection of candidate regulatory polymorphisms for further analyses. The bioinformatics software generated for the analysis has been implemented as a Web-based application system entitled RAVEN (regulatory analysis of variation in enhancers). The RAVEN system is available at http://www.cisreg.ca for all researchers interested in the detection and characterization of regulatory sequence variation.

TP73 allelic expression in human brain and allele frequencies in Alzheimer's disease.

BackgroundThe p73 protein, a paralogue of the p53 tumor suppressor, is essential for normal development and survival of neurons. TP73 is therefore of interest as a candidate gene for Alzheimer's disease (AD) susceptibility. TP73 mRNA is transcribed from three promoters, termed P1 – P3, and there is evidence for an additional complexity in its regulation, namely, a variable allelic expression bias in some human tissues.MethodsWe utilized RT-PCR/RFLP and direct cDNA sequencing to measure allele-specific expression of TP73 mRNA, SNP genotyping to assess genetic associations with AD, and promoter-reporter assays to assess allele-specific TP73 promoter activity.ResultsUsing a coding-neutral BanI polymorphism in TP73 exon 5 as an allelic marker, we found a pronounced allelic expression bias in one adult brain hippocampus, while 3 other brains (two adult; one fetal) showed approximately equal expression from both alleles. In a tri-ethnic elderly population of African-Americans, Caribbean Hispanics and Caucasians, a G/A single nucleotide polymorphism (SNP) at -386 in the TP73 P3 promoter was weakly but significantly associated with AD (crude O.R. for AD given any -386G allele 1.7; C.I. 1.2–2.5; after adjusting for age and education O.R. 1.5; C.I. 1.1–2.3, N= 1191). The frequency of the -386G allele varied by ethnicity and was highest in African-Americans and lowest in Caucasians. No significant differences in basal P3 promoter activity were detected comparing -386G vs. -386A promoter-luciferase constructs in human SK-NSH-N neuroblastoma cells.ConclusionsThere is a reproducible allelic expression bias in mRNA expression from the TP73 gene in some, though not all, adult human brains, and inter-individual variation in regulatory sequences of the TP73 locus may affect susceptibility to AD. However, additional studies will be necessary to exclude genetic admixture as an alternative explanation for the observed associations.

Variations in yeast 3′-processing cis-elements correlate with transcript stability

A large set of yeast mRNA 3′-processing regulatory sequences was analyzed statistically, revealing a systematic variation that correlates with measured mRNA stability. Transcripts with relatively short half-lives have a high frequency of inclusion of 3′-processing elements that include the core sequence of binding sites for the PUF proteins, which enhance mRNA turnover. These results suggest that regulatory sequence variation, typically modeled as random, could arise instead from the necessity or advantage of specifying multiple functions in a common sequence element.

Interelement selection in the regulatory region of the copia retrotransposon.

We report the results of an analysis of naturally occurring cis-regulatory variation within and between two families of the copia Drosophila long terminal repeat (LTR) retrotransposon. The copia 5' LTR and adjacent untranslated leader region (ULR) consists of a number of well-characterized sequence motifs which play a role in regulating expression of the element. In order to understand the evolutionary forces which may be responsible for generating and maintaining copia regulatory sequence variation, we have quantified levels of naturally occurring copia LTR-ULR nucleotide variation and subjected the data to a series of tests of neutrality. Our analysis indicates that the copia LTR-ULR has been subject to negative purifying selection within families and positive adaptive selection between families. We discuss these findings with respect to the regulatory evolution of retrotransposons and the phenomenon of interelement selection.

Evolutionary adaptations of gene structure and expression in natural populations in relation to a changing environment: A multidisciplinary approach to address the million-year saga of a small fish

We have used an experimentally based strategy to address molecular mechanisms underlying adaptation in Fundulus heteroclitus. In an attempt to falsify the hypothesis that selection is a major driving force in the maintenance of genetic diversity, we employed a multidisciplinary approach including allelic isozyme and mtDNA phylogeography, kinetic analyses of allelic isozymes, analysis of variation in coding and regulatory DNA sequences, metabolic biochemistry, organismal physiology, and selection experiments. Observed differences in gene structure and expression led us to make testable predictions about differences in metabolic flux, whole organism performance, and differential survival between allotypes. We have shown that variation in the lactate dehydrogenase-B (Ldh-B) protein results in differences in physiological function and is correlated with differences in survival at high temperatures. Recent work has investigated the role of variation in Ldh-B expression. There are differences in the levels of Ldh-B protein, mRNA, and transcription rate. We have addressed the mechanisms responsible for differences in transcription rate by a combination of sequence comparison, DNase I footprinting, and functional analyses both in vitro and in vivo. We have shown that variation in the regulatory sequence of Ldh-B is responsible for the differences in transcription rate between populations and that the patterns of variation are inconsistent with a neutral model of molecular evolution. This functional differentiation, coupled with departures from neutral expectations, suggests that natural selection has acted on the regulation of Ldh-B. This article illustrates the value of a multidisciplinary approach in addressing problems in gene structure, expression, and evolutionary adaptation.

Evolutionary adaptations of gene structure and expression in natural populations in relation to a changing environment: A multidisciplinary approach to address the million‐year saga of a small fish

We have used an experimentally based strategy to address molecular mechanisms underlying adaptation in Fundulus heteroclitus. In an attempt to falsify the hypothesis that selection is a major driving force in the maintenance of genetic diversity, we employed a multidisciplinary approach including allelic isozyme and mtDNA phylogeography, kinetic analyses of allelic isozymes, analysis of variation in coding and regulatory DNA sequences, metabolic biochemistry, organismal physiology, and selection experiments. Observed differences in gene structure and expression led us to make testable predictions about differences in metabolic flux, whole organism performance, and differential survival between allotypes. We have shown that variation in the lactate dehydrogenase-B (Ldh-B) protein results in differences in physiological function and is correlated with differences in survival at high temperatures. Recent work has investigated the role of variation in Ldh-B expression. There are differences in the levels of Ldh-B protein, mRNA, and transcription rate. We have addressed the mechanisms responsible for differences in transcription rate by a combination of sequence comparison, Dnase I footprinting, and functional analyses both in vitro and in vivo. We have shown that variation in the regulatory sequence of Ldh-B is responsible for the differences in transcription rate between populations and that the patterns of variation are inconsistent with a neutral model of molecular evolution. This functional differentiation, coupled with departures from neutral expectations, suggests that natural selection has acted on the regulation of Ldh-B. This article illustrates the value of a multidisciplinary approach in addressing problems in gene structure, expression, and evolutionary adaptation. J. Exp. Zool. 282:71–94, 1998. © 1998 Wiley-Liss, Inc.