Browsing by Author "Wray, Gregory A"

Increased attention has recently been placed on understanding the natural variation of the malaria parasite Plasmodium vivax across the globe, as in 2020 alone, P. vivax caused an estimated 4.5 million malaria cases and lead to over 600,000 deaths around the world. P. vivax infections in central Africa have been of particular interest, as humans in Sub-Saharan Africa frequently possess a P. vivax resistance allele known as the Duffy-negative phenotype that is believed to prevent infection in these individuals. However, new reports of asymptomatic and symptomatic infections in Duffy-negative individuals in Africa raise the possibility that P. vivax is evolving to evade host resistance.Whole genome sequencing has become more common as a means of understanding the population diversity of P. vivax. However, there is still a scarcity of information about P. vivax in central Africa. In this dissertation, I analyze whole genome sequencing data from a new P. vivax sample collected from the Democratic Republic of the Congo in central Africa. By studying P. vivax from central Africa, we can begin to understand the evolutionary history of the pathogen in this part of the world as it relates to the global context of this pathogen. I also investigate the relationship of P. vivax in the DRC with a potential animal reservoir of a closely related species, P. vivax-like, in non-human primates in this region. Due to the scarcity of P. vivax samples in central Africa, I also investigated methods with which to best make use of whole genome sequencing data, particularly in generating phylogenetic trees. While many studies of P. vivax genetic diversity employ whole genome variation data in order to study evolutionary relationships of P. vivax populations, in this dissertation I make use of the P. vivax apicoplast, a non-photosynthetic plastid organelle genome. The apicoplast genome is five times longer than the mitochondrial genome and does not undergo recombination, making it a valuable locus for studying P. vivax evolutionary history using phylogenetic trees.

Hypersaline-adapted archaea, or haloarchaea, inhabit extreme environments where changes in near-saturated salinity, oxygen, and nutrients require rapid regulation of essential cellular processes. Transcriptional regulatory networks govern the dynamical responses enabling these organisms to sense and respond to rapidly changing conditions. Additionally, timely regulation of carbon metabolic pathways is essential to respond to intermittent nutrient availability and prevent futile cycling of intracellular metabolites. In Halobacterium salinarum, a hypersaline-adapted archaeon that does not rely on carbohydrates for carbon or energy, over 1,500 transcriptome profiling experiments have yielded a genome-scale model of regulatory interactions and revealed a general transcriptional response to diverse stressors (Chapter 2). Among regulators in Hbt. salinarum, a sugar-sensing TrmB family protein has previously been shown to control gluconeogenesis and other biosynthetic pathways. This characterization expanded the set of regulatory functions known for TrmB-family proteins in archaea, which regulate carbohydrate metabolism in hyperthermophilic archaea. TrmB regulators are particularly interesting in Haloarchaea because an expansion of the protein family is presumed to have occurred alongside a diversification of carbohydrate catabolic pathways (Chapter 3).

To investigate whether the expanded set of TrmB functions is shared among haloarchaea with different metabolic capabilities and better understand how regulatory variation arises in extremophiles, we characterized the role of TrmB in two haloarchaeal model species that catabolize carbohydrates: Haloarcula hispanica and Haloferax volcanii (Chapters 4 and 5, respectively). We hypothesized that TrmB would maintain a role in the regulation of gluconeogenesis through homologous targets but would acquire targets involved in the concordant catabolic processes of these saccharolytic models. To characterize the role of TrmB homologs, we conducted high-throughput growth assays, microscopy and other microbiological phenotyping techniques, gene expression profiling via RNA-seq, promoter activity analysis, and protein-DNA binding assays of TrmB homologs in Har. hispanica and Hfx. volcanii. Our results show that TrmB homologs indeed activate gluconeogenesis through the recognition of conserved cis-regulatory motifs. However, contrary to its role in Hbt. salinarum, TrmB does not act as a global regulator in Har. hispanica or Hfx. volcanii: it does not directly repress the expression of peripheral pathways such as cofactor biosynthesis or catabolism of other carbon sources. A key bidirectional control point, activation of ppsA and repression of pyk, is lost in Har. hispanica.

Our results indicate substantial rewiring of the TrmB regulon in Hfx. volcanii. A novel transcriptional regulator, TbsP, is responsible for repressing gluconeogenic genes when glucose is available. TrmB and TbsP appear to compete for partially overlapping binding sites in the promoter of gapII, which encodes the gluconeogenic-specific glyceraldehyde-3-phosphate dehydrogenase. Loss-of-function mutations in tbsP are sufficient to recover partial gapII expression and gluconeogenic activity when trmB is deleted. In Hfx. volcanii, TrmB is predicted to activate ppsA and repress the gene encoding bacterial phosphoenopyruvate carboxylase, perhaps preserving the dynamical and functional behavior of TrmB in Hbt salinarum, but reflecting species-specific anaplerotic strategies. Moreover, TrmB is predicted to repress the expression of bacterial type I GAPDH: gapI and gapII may comprise an additional bidirectional control point in Hfx. volcanii, although this hypothesis requires additional testing.

Cumulatively, this dissertation outlines specific examples of TRN rewiring highlighting the incorporation of metabolic enzymes gained through inter-domain horizontal gene transfer, suggesting rewiring of the TrmB regulon via gain and loss of binding sites alongside metabolic network evolution in Haloarchaea.

In this work, I present four studies across the range of 'omics data types - a Genome- Wide Association Study for gene-by-sex interaction of obesity traits, computational models for transcription start site classification, an assessment of reference-based mapping methods for RNA-Seq data from non-model organisms, and a statistical model for open-platform proteomics data alignment.

Obesity is an increasingly prevalent and severe health concern with a substantial heritable component, and marked sex differences. We sought to determine if the effect of genetic variants also differed by sex by performing a genome-wide association study modeling the effect of genotype-by-sex interaction on obesity phenotypes. Genotype data from individuals in the Framingham Heart Study Offspring cohort were analyzed across five exams. Although no variants showed genome-wide significant gene-by-sex interaction in any individual exam, four polymorphisms displayed a consistent BMI association (P-values .00186 to .00010) across all five exams. These variants were clustered downstream of LYPLAL1, which encodes a lipase/esterase expressed in adipose tissue, a locus previously identified as having sex-specific effects on central obesity. Primary effects in males were in the opposite direction as females and were replicated in Framingham Generation 3. Our data support a sex-influenced association between genetic variation at the LYPLAL1 locus and obesity-related traits.

The application of deep sequencing to map 5' capped transcripts has confirmed the existence of at least two distinct promoter classes in metazoans: focused promot- ers with transcription start sites (TSSs) that occur in a narrowly defined genomic span and dispersed promoters with TSSs that are spread over a larger window. Pre- vious studies have explored the presence of genomic features, such as CpG islands and sequence motifs, in these promoter classes, and our collaborators recently inves- tigated the relationship with chromatin features. It was found that promoter classes are significantly differentiated by nucleosome organization and chromatin structure. Here, we present computational models supporting the stronger contribution of chro- matin features to the definition of dispersed promoters compared to focused start sites. Specifically, dispersed promoters display enrichment for well-positioned nucleosomes downstream of the TSS and a more clearly defined nucleosome free region upstream, while focused promoters have a less organized nucleosome structure, yet higher presence of RNA polymerase II. These differences extend to histone vari- ants (H2A.Z) and marks (H3K4 methylation), as well as insulator binding (such as CTCF), independent of the expression levels of affected genes.

The application of next-generation sequencing technology to gene expression quantification analysis, namely, RNA-Sequencing, has transformed the way in which gene expression studies are conducted and analyzed. These advances are of partic- ular interest to researchers studying non-model organisms, as the need for knowl- edge of sequence information is overcome. De novo assembly methods have gained widespread acceptance in the RNA-Seq community for non-model organisms with no true reference genome or transcriptome. While such methods have tremendous utility, computational complexity is still a significant challenge for organisms with large and complex genomes. Here we present a comparison of four reference-based mapping methods for non-human primate data. We explore mapping efficacy, correlation between computed expression values, and utility for differential expression analyses. We show that reference-based mapping methods indeed have utility in RNA-Seq analysis of mammalian data with no true reference, and that the details of mapping methods should be carefully considered when doing so. We find that shorter seed sequences, allowance of mismatches, and allowance of gapped alignments, in addition to splice junction gaps result in more sensitive alignments of non-human primate RNA-Seq data.

Open-platform proteomics experiments seek to quantify and identify the proteins present in biological samples. Much like differential gene expression analyses, it is often of interest to determine how protein abundance differs in various physiological conditions. Label free LC-MS/MS enables the rapid measurement of thousands of proteins, providing a wealth of peptide intensity information for differential analysis. However, the processing of raw proteomics data poses significant challenges that must be overcome prior to analysis. We specifically address the matching of peptide measurements across samples - an essential pre-processing step in every proteomics experiment. Presented here is a novel method for open-platform proteomics data alignment with the ability to incorporate previously unused aspects of the data, particularly ion mobility drift times and product ion data. Our results suggest that the inclusion of additional data results in higher numbers of more confident matches, without increasing the number of mismatches. We also show that the incorporation of product ion data can improve results dramatically. Based on these results, we argue that the incorporation of ion mobility drift times and product ion information are worthy pursuits. In addition, alignment methods should be flexible enough to utilize all available data, particularly with recent advancements in experimental separation methods. The addition of drift times and/or high energy to alignment methods and accurate mass and time (AMT) tag databases can greatly improve experimenters ability to identify measured peptides, reducing analysis costs and potentially the need to run additional experiments.

The following thesis addresses an issue in ways of knowing that is both commonand destructive in the contemporary American context. Specifically, the issue of misunderstood anthropologies is posited to be an unnecessary destructive force against American churches already in decline. This damage is caused by wooden and polarizing theological and evolutionary anthropologies that underlie the basis of how many define themselves. This project endeavors to show that theological and evolutionary anthropologies are not necessarily adversarial. To this end, the theological anthropology of Gregory of Nyssa as described in On the Making of Man (de Hominis Opificio) and the evolutionary anthropology as described by Charles Darwin in The Descent of Man are defined and compared. These seminal yet still authoritative works are shown to be making different statements about humanity’s coming into being, more so than confrontational ones. The lack of mutual exclusivity between these two anthropologies is heightened by a number of interesting points of connection between them, such as reason being the definitive characteristics of humanity as well as the notion that humanity is continually becoming a more good creature. These ideas will serve to remove barriers of belief for many, all the while providing for a more holistic view of the origins of humanity and thus humanity’s place in the world.

The process by which a single cell develops into a complex multicellular organism with specified cell types and well-defined cellular roles is not completely understood, and the evolution of that process is even more enigmatic. In sea urchin embryos, cell fate specification and differentiation of cell types occurs through gene regulatory networks, or circuits of nodes or genes that interact with and regulate each other’s DNA elements. However, what remains unclear is how a highly conserved developmental GRN can change over evolutionary time and how that can result in altered embryonic form and function.To begin to understand these processes, we developed methods to compare two relatively simple, yet complex sea urchin embryos with radically different life history strategies, the planktotrophic (conserved) Lytechinus variegatus and lecithotrophic (derived) Heliocidaris erythrogramma. To do this, single cell RNA sequencing methods were developed and adapted for the two species to address to what degree the developmental gene regulatory network genes were present, or altered when compared with the known planktotrophic dGRN. Developmental GRN information and gene signatures were applied to assign cell identities, and were vital to the identification of prospective co-expressed candidate dGRN nodes that could participate in a developmental context. With developmental atlases completed in the two species and dGRN nodes examined in both, we then compared gene signatures and dGRN nodes directly to identify prospective candidate dGRN nodes that could participate in the evolution of specification processes. We identified various novel candidates for dGRN analysis and found that, despite the obvious morphological differences, most of the dGRN circuits were conserved. In particular, we found that general specification events are delayed in Heliocidaris erythrogramma; this delay is especially prominent in the skeletogenic precursors, and the number of cells present is greatly reduced—by a factor of 10. In addition, the order of cell specification events, as shown by gene signatures, developmental sub clustering, and integrated analyses, indicate that pigment cells are among the first cell types to be specified, a finding in sharp contrast to known planktotrophic developers. Lastly, this work creates a unified framework of sea urchin development using a novel integrated model based on 1:1 gene orthologs. The application of single cell RNA-seq is a highly useful technique; when applied to a single developmental time series, it can yield insight into the genes and networks deployed over developmental time. Further, when applied to a second developmental time course, it becomes possible to uncover information about the evolution of development, as this application allows us sharp discernment into the genes and networks deployed over evolutionary time.

Although evolution results from differential reproduction and survival at the level of the individual, most research in evolutionary genetics is concerned with comparisons made at the level of divergent populations or species. This is particularly true in work focused on the evolutionary genetics of natural populations. While this level of inquiry is extremely valuable, in order to develop a complete understanding of the evolutionary process we also need to understand how traits evolve within populations, on the level of differences between individuals, and in the context of natural ecological and environmental variation. A major difficulty confronting such work stems from the difficulty of assessing interindividual phenotypic variation and its sources within natural populations. This level of inquiry is, however, the main focus for many long-term field studies. Here, I take advantage of one such field study, centered on the wild baboon population of the Amboseli basin, Kenya, to investigate the possibilities for integrating functional, population, and evolutionary genetic approaches with behavioral, ecological, and environmental data. First, I describe patterns of hybridization and admixture in the Amboseli population, a potentially important component of population structure. Second, I combine field sampling, laboratory measurements of gene expression, and a computational approach to examine the possibility of using allele-specific gene expression as a tool to study functional regulatory variation in natural populations. Finally, I outline an example of how these and other methods can be used to understand the relationship between genetic variation and naturally occurring infection by a malaria-like parasite, Hepatocystis, also in the Amboseli baboons. The results of this work emphasize that developing genetic approaches for nonmodel genetic systems is becoming increasingly feasible, thus opening the door to pursuing such studies in behavioral and ecological model systems that provide a broader framework for genetic results. Integrating behavioral, ecological, and genetic perspectives will allow us to better appreciate the interplay between these different factors, and thus achieve a better understanding of the raw material upon which selection acts.

Many factors, including gene networks, developmental processes, and the environment mediate the link between the activity of genes and complex phenotypes in higher organisms. While genetic variants are the raw material for evolution, these other factors are critical for determining which variants are actually exposed to natural selection. In this dissertation, I describe three projects in which I investigate how developmental mechanisms and the environment interact to shape phenotypic variation. In each project, I use gene expression as a window into the activity of genes, and as a tool to measure variation in and among developmental mechanisms. Two projects are experimental, focusing on early development in sea urchins, and how environmental stress caused by climate change impacts the expression of genetic variation in phenotypic traits. In these projects, I explicitly incorporate information about the biochemical functions of genes and how they interact in development, and test how such mechanisms shape the impact of genetic and environmental perturbations to development. The third project is methodological, in which I propose a unified statistical framework for inferring previously unknown developmental constraints that may underlie gene expression phenotypes. Together, these projects demonstrate that an understanding of developmental mechanisms can enhance our understanding of the processes that shape variation in populations, and can help predict the biological effects of climate change.

In this thesis, I investigate the influence of three different factors (environment, chromatin regulation, and genome structure) on gene expression in the evolution and development of the sea urchin species Heliocidaris erythrogramma and Heliocidaris tuberculata. This species pair share a recent common ancestor but exhibit different life history modes, and thus are an excellent model for studying how evolution acts over (relatively) short time scales to influence phenotype. In Chapter 1, I provide an overview of the research that has been conducted on this species pair over the past several decades. I also survey the literature on gene expression in the context of evolution and development, and discuss the various mechanisms that influence gene expression. In Chapter 2, I evaluate how gene expression in H. erythrogramma is affected by exposure to low-pH seawater, in an effort to quantify how this species may be affected by ocean acidification (OA) caused by anthropogenic climate change. It has been previously shown that OA from seawater uptake of rising carbon dioxide emissions impairs development in marine invertebrates, particularly in calcifying species. Plasticity in gene expression is thought to mediate many of these physiological effects, but how these responses change across life history stages remains unclear. The abbreviated lecithotrophic development of the sea urchin Heliocidaris erythrogramma provides a valuable opportunity to analyze gene expression responses across a wide range of life history stages, including the benthic, post-metamorphic juvenile. I measured the transcriptional response to OA in H. erythrogramma at three stages of the life cycle (embryo, larva, and juvenile) in a controlled breeding design. The results reveal a broad range of strikingly stage-specific impacts of OA on transcription, including changes in the number and identity of affected genes; the magnitude, sign, and variance of their expression response; and the developmental trajectory of expression. The impact of OA on transcription was notably modest in relation to gene expression changes during unperturbed development and dwarfed by genetic contributions from parentage. The latter result suggests that natural populations may provide an extensive genetic reservoir of resilience to OA. Taken together, these results highlight the complexity of the molecular response to OA, its substantial life history stage specificity, and the importance of contextualizing the transcriptional response to pH stress in light of normal development and standing genetic variation to better understand the capacity for marine invertebrates to adapt to OA. In Chapter 3, I examine the regulation of chromatin accessibility, and investigate how the mechanisms that govern regulatory element accessibility are similar to or different from those that govern gene expression. Chromatin accessibility plays an important role in shaping gene expression patterns across development and evolution, but little is known about the genetic and molecular mechanisms that influence chromatin configuration itself. Because cis and trans influences can both theoretically influence the accessibility of the epigenome, I sought to better characterize the role that both mechanisms play in altering chromatin accessibility in H. tuberculata and H. erythrogramma. Using hybrids of the two species, and adapting a statistical framework previously developed for the analysis of cis and trans influences on the transcriptome, I examined how these mechanisms shape the regulatory landscape at three important developmental stages, and compared my results to similar patterns in the transcriptome. I found extensive cis- and trans-based influences on evolutionary changes in chromatin, with cis effects slightly more numerous and larger in effect. Additionally, I found that genetic mechanisms influencing gene expression and chromatin configuration are correlated, but differ in several important ways. Maternal influences also appear to have more of an effect on chromatin accessibility than on gene expression, persisting well past the maternal-to-zygotic transition. Furthermore, chromatin accessibility near GRN genes appears to be regulated differently than the rest of the epigenome, and indicates that trans factors may play an outsized role in the configuration of chromatin near these genes. Together, these results represent the first attempt to quantify cis and trans influences on evolutionary divergence in chromatin configuration in an outbred natural study system, and suggest that the regulation of chromatin is more genetically complex than was previously appreciated. In Chapter 4, I consider how genomic architecture differs in H. tuberculata, H. erythrogramma, and the outgroup species L. variegatus, and how alterations in genome structure may be tied to gene expression differences between these three species. There is not a strong consensus on the connection between genome architecture and evolution, though many studies have related certain elements of genome structural variation to alterations in gene expression. Using chromosome-level genome assemblies and phylogenetic orthology inference, I found that genome-wide synteny is tied to divergence time, and that genes within syntenic regions tend to be conserved in function and copy number compared genes in non-syntenic regions. I also leveraged existing RNA-seq and ATAC-seq datasets for these species to demonstrate that genes in rearranged regions exhibit larger between-species differences in overall gene expression and altered regulatory architecture relative to genes in syntenic regions. Furthermore, in comparing my between-species comparisons of genome architecture, I was able to evaluate the extent to which genomic structural variation might underlie the gene expression differences seen in the transition to lecithotrophy. I found that “isolated” genes (individual orthologs that fall outside of syntenic blocks) exhibit particularly dramatic differences in expression trajectory within the Heliocidaris comparison; moreover, I demonstrate that the large amount of unique sequence in the H. erythrogramma genome harbors regulatory elements that alter gene expression in this species. Together, these results show that the locations of breakpoints regions in this phylogeny are unlikely to be random, and in fact have measurable influences on gene expression and, potentially, life history strategy. In Chapter 5, I contextualize the conclusions from these three studies by considering how I might add to them in the future, and also provide concluding remarks on the impact of my thesis.

The neocortex expanded spectacularly during human origins. That expansion is thought to form the foundation for our cognitive faculties underlying abstract reasoning and socialization. The human neocortex differs from that of other great apes in several notable regards including altered cell cycle, prolonged corticogenesis, and massively increased size. However, despite decades of effort, little progress has been made in uncovering the genetic contributions that underlie these differences that distinguish our species from closely related primate, such as chimpanzees. A subset of highly conserved non-coding regions that show rapid sequence changes along the human lineage are candidate loci for the development and evolution of uniquely human traits. Several studies have identified human-accelerated enhancers, but none have linked an expression difference to a organismal traits, such as brain sizes. Here we report the discovery of a human-accelerated regulatory enhancer (HARE5) near the Wnt receptor FRIZZLED-8 (FZD8). Using a variety of approaches, we demonstrate dramatic differences in human and chimpanzee HARE5 activity, with human HARE5 driving significantly strong expression. We show that HARE5 likely regulates FZD8 and that expression differences influence cell cycle kinetics, cortical layers, and brain size. At present, this would provide the first evidence of a human-chimpanzee genetic difference influencing the evolution of brain size.