Browsing by Subject "DNA methylation"

Now showing 1 - 7 of 7
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    CBD and CpGs: Cannabidiol Neuroepigenetics and a Novel Allele-Specific DNA Methylation Pipeline
    (2023-05) Flack, Nicole
    DNA methylation is an epigenetic mechanism that connects lifestyle and environment to the largely static nucleotide sequence of the genome, influencing function across individual, generational, and evolutionary timescales. Advances in sequencing technology have greatly expanded epigenetic data availability; however, significant technical limitations and knowledge gaps remain. The research presented here interrogates genome-wide differential DNA methylation in three contexts: direct adult cannabidiol (CBD) exposure, developmental CBD exposure, and de novo genome assembly for a non-model species. The objective of the first study was to assess the neuroepigenetic activity, if any, of a popular non-intoxicating phytocannabinoid during direct exposure in adult mice. This work represents the first published exploration of CBD's influence on the brain epigenome; functional analysis of differentially methylated genes revealed changes putatively associated with psychiatric phenotypes. The second study evaluated the consequences of developmental CBD exposure on the adult mouse brain epigenome and behavior. This work revealed persistent, sex-specific behavior changes and perturbed brain DNA methylation in the exposed adult offspring. The third study applied standalone nanopore sequencing for de novo}genome assembly and allele-specific DNA methylation analysis of Pallas's cat (Otocolobus manul), and generated one of the highest-quality reference genomes currently available in Felidae. In the future, this pipeline can be applied to environmental epigenetics experiments to simultaneously obtain genomic and epigenomic data while circumventing several pitfalls of short-read DNA methylation analysis. Collectively, my results illustrate the value of DNA methylation analysis for investigating the functional consequences of a novel exposure, and provide resources for future work in the area of comparative genomics and epigenomics.
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    Characterization of the vasotocin neuropeptide hormone receptor system in the sea lamprey (Petromyzon marinus)
    (2015-11) Mayasich, Sally
    The sea lamprey (Petromyzon marinus) is a jawless vertebrate at an evolutionary nexus between invertebrates and jawed vertebrates. Lampreys are known to possess the arginine vasotocin (AVT) hormone utilized by all non-mammalian vertebrates. We postulated that the lamprey would possess AVT receptor orthologs of predecessors to the arginine vasopressin (AVP)/oxytocin (OXT) family of G protein-coupled receptors found in mammals, providing insights into the early branching into the mammalian V1a, V1b, V2 and OXT receptors. We sequenced one partial and four full-length putative lamprey AVT receptor genes that are found on separate scaffolds in the P. marinus genome. Molecular phylogeny also utilizing the Japanese lamprey (Lethenteron japonicum) genome show that the lamprey receptors cluster with the larger V1a/V1b/OXTR and V2a/b/c clades but specific orthology is unclear. Synteny analysis supports the recently proposed one-round (1R) whole-genome duplication in the vertebrate lineage as the most likely scenario, but does not refute 2R or independent 3R scenarios. The mRNA expression patterns were determined in 15 distinct tissues for these genes, showing transcription in tissues where function has been demonstrated in jawed vertebrates. The literature provides evidence of the expression of neuropeptide hormones and receptors in jawed vertebrate immune cells. For the first time, lamprey peripheral blood leukocytes were maintained in primary culture for periods of at least six days, in which mRNA transcription of a V1a/OXTR-like gene (lamprey AVT receptor Pm807) was demonstrated. These preliminary results also support the hypothesis that neuropeptide hormones may play a role in response to pathogenic challenge in all vertebrates. The possibility of AVT involvement in mediating pheromone release from glandular cells in the gills of mature male lampreys was tested. The compound petromyzonamine disulfate (PADS) was detected at higher quantities after than before injection from several AVT and OXT injected males, but this was not true for the main sex pheromone component 3-keto petromyzonol sulfate (3kPZS). The question of whether DNA methylation of cytosine-guanine (CpG) dinucleotides function to regulate lamprey gene transcription was addressed through analysis of CpG islands in the lamprey Pm807 V1a/OXT receptor gene promoter region. Using High Resolution Melt (HRM) PCR on bisulfite-converted DNA, we pinpointed a region with tissue-specific differences in DNA melt characteristics, indicating differences in methylation level. Sequencing revealed a pattern of methylation at specific CpGs at consistently higher levels in adult heart and larval liver, where Pm807 is transcribed to mRNA, than in adult liver where Pm807 is not transcribed. The methylated CpGs are associated with putative Krüppel-like factor (KLF) 4 transcription factor binding site sequences. In humans KLF4 binds to methylated DNA to initiate transcription. The results suggest that CpG methylation regulates lamprey gene transcription. Additional Pm807 putative promoter elements such as estrogen response element consensus binding sequences were found to be organized similarly to functional OXTR promoters in mammals. The results of my research support the hypothesis that, similarly to jawed vertebrates, differential mRNA expression and resultant functional pleitropy is generated through promoter region sequence and epigenetic regulatory elements in the jawless basal vertebrate lamprey.
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    Connecting variation in genome structure and chromatin composition in Zea mays
    (2021-02) Noshay, Jaclyn
    In many crop species there is tremendous intraspecific variation for genome structure due to highly variable transposon insertions. The goal of my thesis research is to provide insight on genetic and epigenetic dynamics and their relationship with functional variation which has the potential to influence variation in regulation and gene expression. ‘Epigenetics’ describes heritable information not solely due to the DNA sequence, whereas ‘genetics’ is heritable information directly related to the DNA sequence. The central question of the chapters presented is to ask, what are the relative contributions of genetic (transposable element insertions) and epigenetic (localized chromatin changes) factors to variation in DNA methylation and gene expression? In order to address this topic, I first present background information on both DNA methylation and epigenetic influence in maize. It is pertinent to understand the many roles and mechanisms of DNA methylation in plant species in order to decipher the contributions to variation. While this DNA methylation has previously been assessed, the ability to tease apart epigenetics from genetics through polymorphism detection on a genome-wide scale is possible only with new technology. These advancements have helped us to understand information found within the maize epigenome which may not be captured by genetic variation and therefore provide additional data for predicting traits and improving the efficiency of plant improvement strategies. I have conducted several research studies to address the question of epigenetic stability in maize. To first address the dynamic between DNA methylation and transposable elements across the genome, I sought to characterize epigenetic patterns associated with TE families and the cause or effect of TE insertion on DNA methylation architecture.Chapter III presents the assessment of natural variation of transposon insertions and the impact on epiallele state. After identifying how these TEs interact with their flanking sequence I further questioned the genomic influence of the TE body in chapter IV. Accessible chromatin data has allowed identification of putative regulatory regions genome-wide and I pursued the question of how novel TE sequence can contribute to the regulatory dynamics of an organism. Through polymorphic TE insertions we were able to assess the influence of these enhancers on nearby gene expression. The final chapter of my thesis seeks to question the stability of the maize methylome. Now focusing on the shared and nonshared sequence between maize genotypes, I was able to analyze epigenetic variation in the presence or absence of sequence variation. A pan-genome study allows for identification of both core (present across all genotypes) and dispensable (variable between genotypes) epigenetic regions. Presence of variable methylation state is indicative of epigenetic patterns not predictable by sequence. The work presented below describes these broad inquiries in further detail working to answer essential questions regarding genetic and epigenetic contributions to maize phenotype.
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    DEVELOPING CHEMICAL TOOLS TO MAP MOLECULAR MECHANISMS THAT DRIVE DISEASE
    (2023-04) Hurben, Alexander
    Human health is impacted by molecular level events. Within this context, DNA encodes for cellular instructions and proteins execute these genetic protocols to ensure the cell’s components are functioning properly. Thus, the underlying biochemistry within the cell is tightly regulated to ensure vitality. However, when these processes become compromised or damaged, there is increased susceptibility towards developing cancer and neurodegenerative diseases. Often, small chemical changes to our DNA and proteins can be the culprits of such dysregulation. These seemingly minuscule modifications can impair the cell’s proper functions, which can lead to cell death or uncontrolled growth, and ultimately manifest as degenerative disease or cancer. Electrophilic small molecules can be responsible for chemically altering cellular machinery. Additionally, enzymes can make chemical changes such as post-translational modifications on proteins and epigenetic DNA modifications, which can have pronounced effects on cell function and can be equally damaging if not properly regulated. Understanding how molecular events influence health is of paramount importance in designing therapeutics that effectively prevent and treat disease, as well as discovering biomarkers which enable early detection. Despite immense research efforts from the scientific community, there is much remaining to learn about these microscopic processes. This is due to their inherent complexity and lack of technologies to study them. This thesis aims to contribute to addressing this problem by developing new chemical tools which advance our knowledge of the molecular mechanisms that drive disease. This work is composed of seven chapters which explore reactive dopamine metabolites linked to Parkinson’s disease, tools to study the biological implications of elevated intracellular methylglyoxal concentrations, and the development of small molecule epigenetic modulators to regulate aberrant DNA methylation. Chapters I, II, and III of this thesis explores how dysregulated dopamine may contribute to Parkinson’s disease initiation. Chapter I commences with a review of dopamine metabolism and the subsequent generation of reactive dopamine derived metabolites in neurons. This is followed by an overview of protein damaged induced by these metabolites and a review of chemical tools and techniques implemented to study dysregulated dopamine in various experimental systems. Next, Chapter II describes our efforts in designing and implementing a dopamine derived chemical probe to profile dopamine modified proteins which found that dopamine metabolites disrupt protein-folding pathways critical for maintaining healthy neurons. We also detail our development of photoactivatable dopamine probes in Chapter III. Collectively, this work improves the understanding of dopamine protein modification and by extension, molecular events that may contribute to Parkinson’s, which may inform future Parkinson’s therapeutic development. Chapter IV and Chapter V of this thesis focuses on the reactive metabolite methylglyoxal. Methylglyoxal is a sugar-derived metabolite produced naturally in all cells. This reactive compound forms adducts with DNA and proteins, thereby altering their function and influencing cell signalling. Consequently, methylglyoxal protein adducts are implicated in numerous diseases such as cancer, neurodegeneration, diabetes, and cardiovascular disease. In many of these diseases, the cellular processes that break down methylglyoxal become compromised, leading to elevated levels of this reactive molecule within cells. Existing chemical tools to investigate methylglyoxal biology are limited, leading to an incomplete understanding of its physiological and disease-causing roles. Here, we disclose a chemical tool that confers light-mediated release of a methylglyoxal probe within cell models. We use this chemical to identity of the resulting protein adducts. This work enables studying protein adducts induced by methylglyoxal in a controlled fashion to illuminate how this reactive compound impacts various disease states. We also detail our efforts in profiling proteins which undergo covalent DNA crosslinking in the presence of methylglyoxal in Chapter V. This effort is the first study to identify this type of methylglyoxal adduct at a proteome wide scale, which provides a list of candidate methylglyoxal derived DNA-protein cross to investigate in future work. Collectively, these efforts further our understanding of basic methylglyoxal biology and its role in disease progression. Finally, Chapters VI and VII of this thesis describe our efforts towards developing small molecule epigenetic modulators. Regulating gene expression is critical for keeping cells healthy. Over- or under-expressed genes can lead to cancer and other diseases. Accordingly, cells have many methods to control when specific genes are turned on or off in order to function properly; DNA methylation being an example. There are many proteins which control the addition and removal of DNA methyl marks across the genome to ensure appropriate gene expression. Mutations in, or dysfunction of, these proteins can initiate certain cancers. One essential group of proteins involved in removing DNA methylation marks is ten-eleven translocation (TET) methylcytosine dioxygenases (TET). Given TET’s central role in cancer development, theses protein represent a potential drug target. However, there is a paucity of small molecules which selectively inhibit their function without affecting other cellular processes. Thus, there is a need to develop potent and selective compounds which block TET-meditated DNA demethylation. Within Chapter VI, we show our efforts towards the development of novel small molecule TET inhibitors which led us to uncover that copper contamination is responsible for the activity of a reported TET inhibitor. In Chapter VII, we present work on a novel TET inhibitor scaffold which features a bifunctional cofactor-substrate mimetic design. This work has the potential to generate new anticancer therapeutics and improve our understanding of how TET and DNA methylation is linked to cancer development. Ultimately, the work in this thesis provides a novel suite of chemical tools for studying dopamine dysregulation, methylglyoxal metabolism, and TET function. These tools provide insights into cellular damage caused by dopamine and methylglyoxal adducts as well as probes for altering DNA methylation status. Such tools are critical for mapping molecular mechanisms that drive disease.
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    Epigenetic and genetic control of imprinting at the Mez1 locus in maize.
    (2008-05) Haun, WIlliam John
    Genomic imprinting is the mono-allelic expression of gene based on its parent-of-origin and is important for normal progeny development in plants. The goal of this research was to better classify the epigenetic modifications at the Zea mays (maize) imprinted gene Mez1, while also investigating the phenotypic consequence of a loss-of-imprinting. The Mez1 gene in maize is imprinted in endosperm tissue, displaying expression solely from the maternal allele. A differentially methylated region (DMR) was identified in the 5' cis -proximal region of Mez1 in endosperm tissue. In this DMR, the paternal allele displays significantly higher levels of both CpG and CpNpG DNA methylation relative to the corresponding region of the maternal allele. The chromatin modifications of the maternal and paternal alleles of Mez1 and a second imprinted gene, ZmFie1, were studied using allele-specific chromatin immunoprecipitation (ChIP). HistoneH3 and HistoneH4 acetylation are maternally-enriched in endosperm tissue, while HistoneH3 Lysine27 tri-methylation (and to a lesser extent HistoneH3 Lysine27 di-methylation) show paternal allele enrichment. HistoneH3 Lysine9 di-methylation and HistoneH3 Lysine9 tri-methylation do not show parent-specific enrichment. These results suggest DNA methylation and histone modifications are involved in the epigenetic regulation of imprinting in plants. Numerous studies have focused on understanding the mechanism of imprinting, however relatively little is known about the phenotypic consequence of expressing the normally silent allele of an imprinted gene. Several different alleles containing Mu transposon insertions into the 5' cis -proximal region of Mez1 were characterized. Both maternal and paternal inheritance of mez1-mu alleles can result in a loss-of-imprinting. This suggests that Mu transposon insertions at the Mez1 locus can act by disrupting the production of a trans -acting factor or interfering with the cis -acting elements involved in imprinting. Interestingly, the mez1-mu insertions do not effect plant vegetative growth or seed development. These results suggest allelic communication is important between the two parental alleles of imprinted loci.
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    Identification of novel signatures of murine definitive hematopoiesis
    (2014-01) Webber, Beau
    Pluripotent stem cells (PSC) are a tantalizing prospect for a renewable source of patient-specific hematopoietic stem cells (HSC), however efforts to obtain PSC derived HSC capable of long-term engraftment have largely failed. We set out with the primary aim of identifying novel molecular signatures of definitive hematopoiesis, so that these signatures could be applied to improve generation and isolation of HSC in vitro. Toward this end we pursued both discovery and application based strategies centered on Runx1; a transcription factor that is critical for the development of definitive HSC. The discovery arm identified epigenetic modifications at Runx1 cis-regulatory elements that temporally associate with the transition from primitive to definitive hematopoiesis in vivo. We replicated these signatures in vitro by overexpressing HOXB4 in hematopoietic progenitors derived from murine embryonic stem cells (ESC), and found that HOXB4 directly interacts with the definitive-specific distal Runx1 promoter and mediates increased transcription, loss of DNA methylation, and acquisition of active histone modifications at this locus. We next applied our understanding of Runx1 regulation to generate a panel of clonal mESC lines harboring targeted, single-copy fluorescent reporters under the transcriptional control of Runx1 cis-regulatory elements. These lines were used to interrogate the hematopoietic activity of each element independent of copy number and chromosomal position, allowing us to identify combinations that provided optimal activity and fidelity. Building upon this, we established mESC lines harboring synthetic fluorescent and bioluminescent mini genes replicating the structure of the endogenous Runx1 locus and demonstrated that these lines reflect the dynamic promoter switching that occurs at Runx1 during hematogenesis. Sub-fractionation of embryoid body cells based on promoter activity revealed that nearly all colony forming cells (CFC) reside in the distal promoter expressing fraction. With this information we identified specific conditions that could further mature and expand distal positive cells. Collectively, this work identified a previously undescribed molecular signature of definitive hematopoiesis and the mechanism by which it is established. In addition, we applied this knowledge to generate tools with which to interrogate hematopoietic development in vitro, and have demonstrated their utility in optimizing strategies for obtaining definitive hematopoietic progenitors from PSC.
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    Maize bisulfite coupled sequence capture (SeqCap-Epi-v2) probe design
    (2017-12-13) Springer, Nathan M; Li, Qing; Crisp, Peter A; pcrisp@umn.edu; Crisp, Peter A; Springer Lab
    Table detailing the design and genomic coordinates (v2 and v4) for the maize bisulfite coupled sequence capture (SeqCap-Epi-v2) probe design for profiling of the maize methylome. Released to accompany our publication on DNA methylation changes induced by tissue culture (Han et al 2017) as a supporting supplemental methods file.