1,721,057 research outputs found
Studies into Location-specific cis-Regulatory Motifs
No full textGene expression and regulation are major determinants of phenotypic traits displayed across species. Although the DNA sequence elements that control gene expression play a crucial role in determining species morphology, predicting cis-regulatory elements through sequence analysis alone remains a difficult task. A few regulatory elements, such as the TATA-box and Initiator sequence, have been known to exhibit overrepresentation at specific locations within the proximal promoter. However, the extent to which this occurs among cis-regulatory elements is not well understood. Here, we take a genome-wide approach towards detecting such functional sequence elements, using location-specific overrepresentation as a criterion for regulatory function. We provide evidence that a surprisingly large number of regulatory elements exhibit locational overrepresentation with respect to the transcription start site. We then utilize this characteristic to predict novel cis-regulatory elements overrepresented at particular locations within the proximal promoter.Transcriptional regulation is most often controlled not by single protein factors acting in isolation, but instead multiple transcription factors acting together within multi-protein complexes. As protein-protein interactions are largely determined through protein structure, we would expect to see patterns of spatial preference between motif-pairs binding interacting factors. However, in the absence of methods to predict such spatial preferences between motifs, comprehensive assessments of such inter-relationships have not been previously conducted. As our model provides a general tool for detecting positional specificities of a motif relative to a given reference point, we expanded our model to measure distance preferences between pairs of motifs on a genome-wide scale. We show that there often exist patterns of spatial dependencies between pairs of sequence elements that bind interacting protein factors. We find that regulatory motifs binding interacting proteins often have multiple inter-motif distances at which they preferentially occur, and we show that the intervals between preferred distances are highly consistent across motif-pairs. This distance preference `phasing' was empirically found to occur at consistent intervals around ~8-10 bp, corresponding to approximately the number of nucleotides within a single turn of the DNA double-helix. This finding suggests a tendency for protein factor-pairs to interact in a specific orientation with respect to the turn of the DNA molecule, and offers a convenient method by which to determine motif-pairs binding interacting transcription factors de novo. While little is known about the mechanisms by which individual cis-regulatory elements ultimately control gene expression, even less is known about how such elements evolve over time. A single transcription factor can potentially target hundreds of genes across the genome, and thus modifications in the binding affinities of such proteins must induce conversions at a multitude of functional sites in order to preserve the set of target genes that the trans-factor regulates. It is therefore commonly assumed that such changes occur rarely and at a slow rate over the course of evolution. Despite this widespread assumption, we find that a surprisingly large number of cis-regulatory elements have been subject to significant changes in consensus sequence in a lineage-specific manner. Here, we demonstrate that the genomic landscape is highly adaptable, rapidly adjusting to global changes in preferred regulatory consensus sequences. Focusing upon regulatory elements exhibiting location-specific overrepresentation, we find that a substantial fraction of regulatory elements have been subject to evolutionary modifications, even between closely related eutherians. These findings have broad implications regarding evolving phenotypes observed across species.</p
The Evolution of Adipocytes During Human Origins: An Integrative Genomic, Cellular, and Molecular Approach
No full textUntil recently, understanding of the evolutionary and molecular basis of many uniquely human traits has largely been hampered by the limited ability to perform experiments on, and access to samples from non-human primates. Induced pluripotent stem cell (iPSC) lines have provided virtually unlimited samples and allow, for the first time, precise experimental control in order to understand the relative roles of genetic, epigenetic and environmental effects. Selective forces have shaped our species throughout our evolutionary history, and by understanding how these forces work, we can gain valuable insight into how we are influenced by our environment. More specifically, by understanding how the traits that make us uniquely human evolved, we can begin to learn about the benefits, tradeoffs, and environmental interactions of each trait in the context of our survival and health. Traits that today are maladaptive may have been beneficial to us in the past when we were living in different environmental conditions.This dissertation focuses on adipocytes, the key component cell of fat, whose function was crucial during human evolutionary origins, as metabolic traits and diet were of particular importance. We have utilized of iPSCs from humans and chimpanzees to examine genome-wide gene expression and regulatory-element activity as well as perform experimental manipulation. Comparisons of human and chimpanzee adipocytes has allowed for the identification of species-specific differences in gene expression and open chromatin regions. We find enrichment in several interesting categories including ‘energy homeostasis, ‘diacylglycerol metabolism’ and ‘carnitine biosynthetic process’. We identify several genes related to the processing of dietary fatty acids that are expressed at a higher level in human adipocytes (FADS1/2 and ACSL5/6). Furthermore, we show that human lipid droplets are larger than chimpanzee droplets in white adipocytes but not brown adipocytes. Together this suggests concerted differences in how humans process and store dietary fatty acids.</p
Developmental Single-Cell RNA Sequencing in the Sea Urchin Species Lytechinus variegatus and Heliocidaris erythrogramma
No full textThe 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.</p
An evolutionary genomics approach towards understanding Plasmodium vivax in central Africa
No full textIncreased 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.</p
Selection and Constraint: Population Genetic Approaches to Understanding the Evolution of Sea Urchin Development
No full textChanges in the expression and function of genes active during metazoan development have played a critical role in the evolution of morphological differences between species and phyla, yet the origins of these changes remain poorly understood. What roles do positive and negative selection play in the evolution of development? How do evolutionary changes accumulate given the degree to which organisms are able to buffer the effects of environmental and genetic perturbations during development? The crucial insight of the Modern Evolutionary Synthesis was that divergence between species arises from variation within populations. Following this principle, I have made use of tools from quantitative and population genetics to investigate three central questions: 1) How much genetic variation is there in the networks of genes that underlie metazoan development? 2) What affect does developmental buffering have on the accumulation of selectable genetic variation? 3) To what extent does selection act to shape patterns of genetic variation among different kinds of genes and at different stages of development? I show that developmental systems can harbor extensive levels of genetic variation, and that the amount of genetic variation in individual genes at different stages of development is related to the extent to which variation in those genes is buffered by genetic interactions. I also show that while selection plays an active role in shaping genetic variation in development, the extent to which variation in a gene is visible to selection depends in predictable ways on a) the biological function of that gene and b) whether the mutations in question influence gene expression or protein function. My results as a whole demonstrate the utility of population level approaches to the study of the evolution of development, and provide key insights into the role that selection plays in generating developmental variation.</p
Genetic and Environmental Constraints on Developmental Systems: Towards Predicting Genetic Responses to Climate Change in Sea Urchins
No full textMany 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.</p
Genomic Basis for a Developmental Life History Switch in the Sea Urchin Heliocidaris erythrogramma
No full textLecithotrophic (non-feeding) larval development has independently evolved numerous times in marine invertebrates from an ancestral, planktotrophic (feeding) larval state. The evolution of this developmental mode in a species is accompanied by dramatic changes in ecology and development, including lower fecundity, higher maternal investment per offspring, changes in egg composition, alteration of embryonic fate specification, morphologically simple larvae, and reduced time to metamorphosis. Thus, the evolutionary switch between lecithotrophy and planktotrophy serves as an exemplary system for investigating the effect of changing ecological pressures on the evolution of novel developmental phenotypes. The sea urchin genus Heliocidaris represents one of the best studied examples of this switch, in which H. erythrogramma evolved lecithotrophy around five million years ago. Over the past several decades, previous work has documented phenotypes distinguishing development of this species from the ancestral, planktotrophic condition. These phenotypes range from increased sperm size and hypertrophy of lipid deposition in the egg, to changes in embryonic axis determination, delayed blastomere specification, and alterations to spatial and temporal expression of key developmental network genes. Although much is known about what phenotypes are associated with the evolution of lecithotrophy in this species, much less is known of the regulatory mechanisms for how these changes arose in the first place. This gap in knowledge is the subject of my thesis: to gain a better understanding of the genomic and molecular basis for the evolution of lecithotrophy in H. erythrogramma.
To accomplish this, I carried out a set of physiological and genomic comparisons between H. erythrogramma, a closely-related planktotrophic congener H. tuberculata, and a distantly-related planktotroph Lytechinus variegatus in order to identify specific molecules and genomic loci underlying lecithotrophic development. In Chapter One, I analyzed lipid and protein content of eggs and larvae from these three species using mass spectrometry to characterize metabolic differences in egg provisioning and embryogenesis in H. erythrogramma. In Chapter 2, I present a chromosome-level assembly of L. variegatus, highlighting a genome assembly and annotation method that will be applied to the two Heliocidaris species and the utility of a high-quality genome assembly for functional genomic analysis. In Chapter 3, I compare the genome assemblies of H. erythrogramma and H tuberculata to show that a conserved developmental network controlling sea urchin development has been dramatically modified in H. erythrogramma through genic and non-coding modifications. In Chapter 4, I compare the chromatin landscapes of these three species through development using ATAC-seq to access how cis-regulatory mechanisms have evolved during the acquisition of lecithotrophic development.
From this work, I found that the enormous lipid provisioning of H. erythrogramma eggs is composed primarily of diacylglycerol ether lipids and that these lipids are not metabolized for pre-metamorphic development, but instead provisioned to promote post-metamorphic survivorship of juvenile individuals. Instead, upregulated glycolysis proteins suggest this pathway may be driving rapid pre-metamorphic development. Comparative genomic analyses demonstrate positive selection and changes to chromatin accessibility have modified the regulatory genome of H. erythrogramma, especially near developmental network genes, and that these changes are associated with temporal and spatial differences in embryonic gene expression. Furthermore, the Pmar1 transcription factor family has likely lost its ancestral function in specifying the primary mesenchyme lineage in this species, a cell type responsible for larval skeletal development and patterning of the embryo. Finally, development has one of the largest effects on changes in chromatin accessibility in each species, but particularly near developmental genes, embryonic chromatin dynamics is highly associated with the life history strategy of each species. Future work identifying examples of convergent or novel pathways driving evolution of lecithotrophy in other echinoids will provide valuable insight into general principles governing how derived developmental phenotypes can evolve at short evolutionary timescales.
</p
Leveraging Complementary In Vivo and In Vitro Gene Expression Measurements to Elucidate Uniquely Human Metabolic Processes
No full textThe origin of man has motivated researchers to investigate differences between humans and our non-human relatives. The striking phenotypic differences that distinguish humans from chimpanzees are likely controlled by a relatively modest number of genetic changes present between these species. As energy acquisition and processing effect multiple organ systems, the dramatic changes in the human diet are thought to underpin many of these unique phenotypes. The evolution of the human diet is marked by omnivory with increased consumption of animal products, cereal grain and vegetable oil associated with the Paleolithic era, domestication of plants and the industrial revolution respectively. Nutrition is essential for life and is unique as it both shapes, and is shaped by the genome. Given this complex interaction, teasing out actors and responders in the genome-diet relationship is a challenge. I took several expression approaches by interrogating regulatory regions, candidate networks and genomes in tissues of dietary relevance. These experiments uncovered combinations of physiological and morphological changes between humans and non-human primates. Taken together, the combined power of in vitro and in vivo approaches elucidates several genetic mechanisms important in uniquely human bioenergetic processes.</p
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
- …
