Understanding Large Physiological Variation: Genomics, Genes, and Gene Expression

Studies investigating genotype to phenotype relationships have demonstrated that genetic variation is biologically important; however, the evolutionary significance of these relationships and variation maintenance remains poorly understood. Here, three wild populations of the small teleost fish Fundulus heteroclitus: one locally heated by thermal effluent from a nuclear power plant (TE) and two reference populations that do not experience local heating (10 km north and 3.5 km south) were used. This experimental design was intended to test the central hypothesis that the TE population is locally adapted when compared to the north and south reference sites. Temperature specific metabolic and thermal tolerance traits, tissue specific (heart and brain) mRNA expression, and genetic variation (single nucleotide polymorphisms, SNPs) are compared among populations. Few differences exist among populations in physiological traits, mRNA expression, or SNPs. Instead, large inter-individual variation in metabolic and thermal tolerance traits is irrespective of population. This large variation is explained by tissue and acclimation temperature specific mRNA expression patterns, with different mRNAs in heart and brain tissue from similar pathways explaining trait variation. Whole genome sequencing for the same individuals that physiological traits and mRNA expression were measured in allowed direct (genome wide association, GWAS) and indirect (expression quantitative trait loci, eQTL) associations with physiological traits to be examined. Many significant eQTL explain single and multivariate mRNA expression patterns. These tissue specific eQTL are dominated by trans-acting factors (found on different chromosomes than the mRNAs they impact). This thesis contributes new knowledge on the evolutionary importance of biological variation.&nbsp;</p

Paradoxical whole genome DNA methylation dynamics of 5’aza-deoxycytidine in chronic low-dose exposure in mice

Decitabine (5-aza-2ʹdeoxycytidine; DAC) is a DNA methyltransferase inhibitor used to hypomethylate the epigenome. Current dosing regimens of DAC for use in mice vary widely and their hypomethylating ability has not been robustly characterized, despite reliable results of hypomethylation of the epigenome with cell lines in vitro and tissue specificity in vivo. We investigated the effects on the DNA methylome and gene expression within mice exposed to chronic low doses of DAC ranging from 0 to 0.35 mg/kg over a period of 7 weeks without causing toxicity. Our dose paradigm resulted in no cytotoxic effects within target tissues, although testes weight and sperm concentration significantly reduced as dose increased (p-value <0.05). By whole genome bisulphite sequencing (WGBS), we identify tissue and dose-specific differentially methylated CpGs (DMCs) and regions (DMRs) in testes and liver. Testes methylation is more sensitive to DAC exposure when compared to liver, cortex, and hippocampus. Gene expression was dysregulated in testes and liver, targeting non-specific pathways as dose increases. Together our data suggest DNA methylation and gene expression are disrupted by in vivo DAC treatment in a non-uniform manner contrary to expectations, and that no dose level or regimen is sufficient to cause systemic hypomethylation in whole mice

Trans-Acting Genotypes Associted with mRNA Expression Affect Metabolic and Thermal Tolerance Traits

Abstract Evolutionary processes driving physiological trait variation depend on the underlying genomic mechanisms. Evolution of these mechanisms depends on the genetic complexity (involving many genes) and how gene expression impacting the traits is converted to phenotype. Yet, genomic mechanisms that impact physiological traits are diverse and context dependent (e.g., vary by environment, tissues), making them difficult to discern. We examine the relationships between genotype, mRNA expression, and physiological traits to discern the genetic complexity and whether the gene expression affecting the physiological traits is primarily cis or trans-acting. We use low-coverage whole genome sequencing and heart- or brain-specific mRNA expression to identify polymorphisms directly associated with physiological traits and expressed quantitative trait loci (eQTL) indirectly associated with variation in six temperature specific physiological traits (standard metabolic rate, thermal tolerance, and four substrate specific cardiac metabolic rates). Focusing on a select set of mRNAs belonging to co-expression modules that explain up to 82% of temperature specific traits, we identified hundreds of significant eQTL for mRNA whose expression affects physiological traits. Surprisingly, most eQTL (97.4% for heart and 96.7% for brain) were trans-acting. This could be due to higher effect size of trans versus cis-acting eQTL for mRNAs that are central to co-expression modules. That is, we may have enhanced the identification of trans-acting factors by looking for SNPs associated with mRNAs in co-expression modules that broadly influence gene expression patterns. Overall, these data indicate that the genomic mechanism driving physiological variation across environments is driven by trans-acting heart- or brain-specific mRNA expression

Transcriptomic analysis provides insights into molecular mechanisms of thermal physiology

Physiological trait variation underlies health, responses to global climate change, and ecological performance. Yet, most physiological traits are complex, and we have little understanding of the genes and genomic architectures that define their variation. To provide insight into the genetic architecture of physiological processes, we related physiological traits to heart and brain mRNA expression using a weighted gene co-expression network analysis. mRNA expression was used to explain variation in six physiological traits (whole animal metabolism (WAM), critical thermal maximum (CT), and four substrate specific cardiac metabolic rates (CaM)) under 12 °C and 28 °C acclimation conditions. Notably, the physiological trait variations among the three geographically close (within 15 km) and genetically similar F. heteroclitus populations are similar to those found among 77 aquatic species spanning 15-20° of latitude (~ 2,000 km). These large physiological trait variations among genetically similar individuals provide a powerful approach to determine the relationship between mRNA expression and heritable fitness related traits unconfounded by interspecific differences. Expression patterns explained up to 82% of metabolic trait variation and were enriched for multiple signaling pathways known to impact metabolic and thermal tolerance (e.g., AMPK, PPAR, mTOR, FoxO, and MAPK) but also contained several unexpected pathways (e.g., apoptosis, cellular senescence), suggesting that physiological trait variation is affected by many diverse genes

Evolutionary conservation of DNA methylation in CpG sites within ultraconserved noncoding elements

<p>Ultraconserved noncoding elements (UCNEs) constitute less than 1 Mb of vertebrate genomes and are impervious to accumulating mutations. About 4000 UCNEs exist in vertebrate genomes, each at least 200 nucleotides in length, sharing greater than 95% sequence identity between human and chicken. Despite extreme sequence conservation over 400 million years of vertebrate evolution, we show both ordered interspecies and within-species interindividual variation in DNA methylation in these regions. Here, we surveyed UCNEs with high CpG density in 56 species finding half to be intermediately methylated and the remaining near 0% or 100%. Intermediately methylated UCNEs displayed a greater range of methylation between mouse tissues. In a human population, most UCNEs showed greater variation than the LINE1 transposon, a frequently used epigenetic biomarker. Global methylation was found to be inversely correlated to hydroxymethylation across 60 vertebrates. Within UCNEs, DNA methylation is flexible, conserved between related species, and relaxed from the underlying sequence selection pressure, while remaining heritable through speciation.</p

Variation Among Fundulus heteroclitus Genomic Regions in CpG Site Density and Implications for Environmental Epigenetics

Academi

Measuring complex phenotypes: A flexible high-throughput design for micro-respirometry

Abstract Variation in tissue-specific metabolism between species and among individuals is thought to be adaptively important; however, understanding this evolutionary relationship requires reliably measuring this trait in many individuals. In most higher organisms, tissue specificity is important because different organs (heart, brain, liver, muscle) have unique ecologically adaptive roles. Current technology and methodology for measuring tissue-specific metabolism is costly and limited by throughput capacity and efficiency. Presented here is the design for a flexible and cost-effective high-throughput micro-respirometer (HTMR) optimized to measure small biological samples. To verify precision and accuracy, substrate specific metabolism was measured in heart ventricles isolated from a small teleost, Fundulus heteroclitus , and in yeast ( Saccharomyces cerevisiae ). Within the system, results were reproducible between chambers and over time with both teleost hearts and yeast. Additionally, metabolic rates and allometric scaling relationships in Fundulus agree with previously published data measured with lower-throughput equipment. This design reduces cost, but still provides an accurate measure of metabolism in small biological samples. This will allow for high-throughput measurement of tissue metabolism that can enhance understanding of the adaptive importance of complex metabolic traits

An Innovative Setup for High-Throughput Respirometry of Small Aquatic Animals

Metabolic rate is often measured as a phenotype in evolutionary genetics, among other fields including many facets of physiology, behavior, and ecology, because it impacts organismal fitness, is repeatable and heritable, and is responsive to numerous environmental variables. Aquatic respirometry, a method used to measure metabolic rate, has allowed key questions in these fields to be investigated, namely: (1) why do individuals from the same population exhibit up to 3-fold differences in metabolic rate, (2) how does metabolic rate change during an individual’s lifetime, and (3) what metabolic rate is advantageous in a specific environment? Current respirometry studies often suffer from small sample sizes and rely on low throughput approaches to measure metabolic rate, making it difficult to answer these and other relevant ecological and evolutionary questions due to lack of power, failure to capture true biological variation, and confounding variables, like time, that are introduced due to limitations in methodology. Here we describe a scalable high-throughput intermittent flow respirometer (HIFR) design and use it to measure the metabolic rates of 19 aquatic animals in one night while reducing equipment costs and time by more than 50%