Browsing by Subject "epigenetics"

Now showing 1 - 5 of 5
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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 and Classifcation of HDACs in Aging Osteoclasts
    (2022-05) Schleyer, Brendan
    Background: As humans age, bone mass is lost due to increased osteoclast and decreased osteoblast function. Bone cell differentiation is regulated by epigenetic changes within histones through acetylation/ de-acetylation. Histone deacetylase removes an acetyl group from a histone, repressing transcription. Several studies have demonstrated that loss of HDAC expression enhances osteoclast activity. There are 18 currently identified HDACs which are divided in 3 classes: I, II, and IV. This study aims to examine expression levels of HDAC class I and II in osteoclasts at 1 and 24-months of age. We hypothesize that osteoclasts from older mice will exhibit lower HDAC expression. This will increase our understanding of how HDAC expression changes in osteoclasts from aging mice. These changes may suggest a possible mechanism by which osteoclast activity is increased in aging osteoclasts. Methods: Bone marrow cells were flushed from femurs and tibiae of either male or female 1- or 24-month mice. BMMs were harvested and differentiated into osteoclasts at days 0, 2, and 4. They were then lysed to isolate RNA and reverse transcriptase was added to yield cDNA. Samples were subjected to qRT-PCR. Data analysis yielded expression coefficients with standard deviations. True expression was calculated for data sets and examined in graph form showing average with +/- standard deviation. Multiple group comparison ANOVA tests were run with significance set at p< 0.05. Results: For HDAC 4, expression at day 4 of differentiation of 24-month females was significantly higher than the 1-month females (p= 0.0273). For HDAC 11, expression at day 4 of differentiation of 1-month males was significantly higher than that of the 24-month males. No other group comparison yielded significance. Overall, expression was similar between age groups and sexes. Expression levels were shown to differ between days of differentiation. Conclusions: This study functions as a pilot study regarding HDAC classification and expression. To the knowledge of the author, there are no studies to date examining HDAC expression between young and advanced age subjects. The acquired data has numerous outliers which may be disguising areas of significance. The data does not support our hypothesis that expression is lower in advanced age subjects. Limitations of the study include number of test subjects, quality/quantity of cDNA, and accuracy of sample preparation. On a broader scope, the data does not depict distinct expression patterns for the varying classes of HDACs. It is suggested that each HDAC has varying expression levels at different days of differentiation and might have roles at various stages during expression. This study provides a groundwork for moving forward with more targeted studies based on conditions such as osteoporosis and periodontitis.
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    Development of Small-Molecule Inhibitors and Biophysical Tools to Study the Epigenetic Protein BPTF
    (2022-06) Zahid, Huda
    Bromodomains are protein-protein interaction modules involved in epigenetic regulation of gene expression, typically through the recognition of acetylated lysine residues in histones. Bromodomain-containing proteins are involved in several disease processes, including cancer, inflammation and viral replication. There are 61 known human bromodomains and due to their high structural similarity, developing small-molecule inhibitors that bind with high affinity to specific proteins is a major challenge in the field. While numerous small molecule probes have been developed for the BET (bromodomain and extra terminal) family, few have been reported for the 53 non-BET bromodomains. Potent and selective chemical probes are, therefore, required to enhance our biological understanding of the non-BET bromodomain family. Among them, the understudied protein Bromodomain and PHD finger Transcription Factor (BPTF) is known to play an important role in chromatin remodeling and is the focus of this dissertation. BPTF is overexpressed in several cancers such as melanoma, breast cancer and high-grade gliomas. It has been identified as a potential target for developing cancer therapeutics and BPTF bromodomain inhibitors are being explored for combination treatment with chemotherapeutics. Given the emerging significance of BPTF as an anticancer target, small-molecule inhibitors for this bromodomain-containing protein are needed. This dissertation details efforts to develop potent chemical inhibitors of the BPTF bromodomain through systematic structure-activity relationship analyses. Starting from a reported pyridazinone scaffold and using structure-based design approaches, the lead compound BZ1 is developed with a Kd of 6.3 nM for BPTF, making it one of the most potent inhibitors reported to date for this bromodomain. These efforts are assisted by the optimization of a bead-based competition assay AlphaScreen (described in Chapter 2) which is used to quantify affinity values and cross-validate other biophysical methods for studying BPTF interactions. Several of the first cocrystal structures of BPTF bound to small-molecule ligands are also reported, which are key to rational design of potential drug candidates. We further show that these compounds sensitize breast cancer cells to the chemotherapeutic doxorubicin, indicating that inhibition of the BPTF bromodomain can overcome chemoresistance in these cells. Efforts are now underway to evaluate the drug-like properties of pyridazinone inhibitors for in vivo studies. The development of potent BPTF inhibitors paved the way to heterobifunctional molecules described in Chapter 4 of this dissertation. In this study, degraders based on a pyridazinone scaffold are designed and evaluated using an in-cell NanoBRET assay. As first-generation degraders, these compounds induce ternary complex formation of the BPTF bromodomain with the cereblon E3 ligase, evident through both in vitro and in-cell assay formats. Using an optimized NanoBRET assay, we show that they also efficiently degrade a Nanoluciferase-BPTF bromodomain construct. While preliminary western blotting studies indicate that these pyridazinone-based degraders do not show any degradation activity for endogenous BPTF, future work will look at other cell lines and various linkers to optimize the degradation efficiency of these compounds. In another project, detailed in Chapter 5, a fundamental study of noncovalent and highly stabilizing sulfoxide-aromatic interactions in proteins is described. This will provide insight into the effects of oxidation of methionine residues in α-synuclein, a modification known to be involved in neurodegenerative disorders such as Parkinson’s disease. β-hairpin peptides are used as model systems to study these motifs and quantify the interaction of oxidized methionine with aromatic side chains of amino acids. In this system, no additional stabilization is observed when interactions of phenylalanine with methionine and oxidized methionine are compared. Further analysis with tryptophan indicates a slight destabilization of the β-hairpin peptide motif with oxomethionine. Follow-up studies for this project will look at other aromatic side chains such as tyrosine and the application of this approach in the broader context of α-synuclein.
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    Epigenetic Mechanisms in Lung Cancer
    (2018-09) Seiler, Christopher
    Epigenetic control of gene expression involves covalent reversible modifications of DNA, RNA, and histones which lead to changes in chromatin structure and accessibility. The ability to maintain precise control over gene expression in cells and tissues is critical for ensuring normal cellular development and homeostasis. The most important epigenetic mark of DNA is methylation of cytosine at the C5 position (MeC). This stable epigenetic mark is introduced by de novo methyltransferases DNMT3a/b and maintained through cell division by maintenance methyltransferase DNMT1. Ten Eleven Translocation (TET) dioxygenases oxidize 5-methylcytosine (MeC) to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxylcytosine (caC), a process known to induce DNA demethylation and gene reactivation. A precise balance of DNA methylation and demethylation is important for establishing tissue specific gene expression patterns, maintaining cell identity, and guiding development. However, inflammation and exposure to exogenous agents can lead to changes in DNA methylation patterns or “epimutations” which together with genetic mutations can lead to the development of cancer. Chapter I of this Thesis provides an overview of the major mechanisms of epigenetic regulation including epigenetic marks of DNA, non-coding RNAs, and histone modifications. Chapter I then describes epigenetic dysregulation in cancer and other human diseases. We then go on to describe how epigenetic changes in DNA can be detected and quantified using antibodies and mass spectrometry-based approaches. After considering global quantitation of epigenetic DNA modifications, we discuss the methods available for mapping epigenetic modifications along the genome. In Chapter II of this thesis, the effects of C5-cytosine substituents with increased steric bulk on catalytic activity of maintenance DNA methyltransferase (DNMT1) were examined. This protein specifically recognizes 5-methylcytosine (MeC) bases at hemimethylated CG sites in DNA and conducts maintenance methylation. Maintenance methyltransferase activity was the highest towards DNA containing the natural DNMMT1 substrate, MeC. The enzyme was capable of performing maintenance methylation when 5-ethyl-dC was the substrate, while the more rigid and bulky C5-alkyl substituents such as 5-vinyl- dC, and 5-propyl-dC could not direct maintenance methylation. In Chapter III, we investigated the kinetics of maintenance DNA methylation towards DNA duplexes containing oxidized forms of MeC (hmC, fC, and caC). We also employed a molecular dynamics simulation of the enzyme with the DNA to understand the interactions of oxidized forms of MeC with the DNMT1 enzyme. We found that methyl transfer rates were reduced when MeC was oxidized to hmC, fC, and caC, consistent with the model that Tet mediated oxidation contributes to passive DNA demethylation. In Chapters IV and V, we investigated inflammation-mediated epigenetic changes in the lung using A/J mouse model of smoking induced lung cancer. In collaboration with NuGEN (Santa Carlos, CA), we developed a novel reduced representation bisulfite sequencing (RRBS) methodology to map both MeC and hmC genome-wide. Our studies provide evidence that inflammation of the lung induces both global and site-specific epigenetic changes in DNA methylation and hydroxymethylation, alters global histone acetylation, and deregulates gene expression. These studies also provide evidence that exposure to cigarette smoke can alter site-specific DNA methylation and hydroxymethylation of genes that are associated with the cancer phenotype. The final chapter of this dissertation (Chapter VI) employs affinity proteomics to identify protein readers of epigenetic marks of DNA in the lung. DNA duplexes functionalized with C, MeC, hmC, fC, and caC were attached to solid support and incubated with nuclear protein extracts from human bronchial epithelial cells (HBEC). Proteins specifically recognizing DNA epigenetic marks were identified using Orbitrap Velos mass spectrometer and quantified using 8-plex TMT tags. This chapter details the development of a method for carrying out the affinity proteomics experiments, including solid phase synthesis of DNA targets, peptide tagging, sample clean-up, fractionation, and nanoHPLC-ESI+-MS2 based methodology for protein identification and quantification. Overall, during the course of the studies described in this thesis, we have investigated the specificity and kinetics of human maintenance DNA methyltransferase (DNMT1), employed animal models to characterize epigenetic changes in the lung caused by inflammation and exposure to cigarette smoke, and examined novel mechanisms of epigenetic regulation at oxidized forms of MeC. Overall, this work contributes to current understanding of epigenetic regulation in normal cells and epigenetic deregulation in cancer.
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    Epigenetic regulatory mechanisms that govern cardiovascular development
    (2022-06) Sierra Pagan, Javier
    Cardiovascular disease (CVD) remains the number one cause of death in the United States and the World. The clinical outcomes of patients with heart failure, a form of CVD, remain poor because current clinical therapies do not address a critical feature of heart failure which is the loss of functional cardiac muscle. To decrease the morbidity and mortality in these patients, several strategies are being developed to replace the loss of functional cardiac muscle with new one. Two attractive strategies for treating CVD involve converting cardiac fibroblasts (reprogramming) into functional muscle or vascular cells and promoting cell cycle re-entry of adult cardiomyocytes following cardiac injury to replace the dead muscle. While the adult mammalian heart has limited regenerative potential following injury, the embryonic and neonatal mammalian heart has a remarkable regenerative capacity. Therefore, our goal for these studies was to define new factors and mechanisms that could enhance repair and regeneration in the adult mammalian heart. To this end, in this thesis, we identified novel epigenetic regulatory mechanisms that govern cardiovascular development, particularly within the vascular and cardiac muscle lineages. Our first finding is that we identified that ETV2 functions as a pioneer transcription factor that relaxes closed chromatin and regulates endothelial development. We did this by comparing engineered embryonic stem cell (mESCs) differentiation and reprogramming models (MEFs) with multi-omics techniques, we demonstrated that ETV2 was able to bind nucleosomal DNA and recruit BRG1. The recruitment of BRG1 led to the remodeling of chromatin around endothelial genes and helped to maintain an open configuration, resulting in increased H3K27ac deposition. Our second finding is that we discovered a signaling cascade where ETV2 regulates RHOJ expression during endothelial progenitor cell migration. We did this by combining computational genomics (RNAseq, ATACseq and ChIPseq) to discover that ETV2 regulates migratory pathways through the expression of RHOJ, particularly in developing endothelial progenitor cells (E7.75 and E8.5 mouse embryos and developing mESCs). Our third finding is that we identified FOXK1 as an essential transcriptional and epigenetic regulator of cardiovascular development. We used mESCs that lacked FOXK1 expression and discovered that in its absence, cardiac muscle development is significantly affected, both at the transcriptional and chromatin level. Mechanistically, we also discovered that FOXK1 represses Wnt signaling, particularly Wnt6, to promote the development of cardiac progenitor cells. ETV2 has the capacity to reprogram fibroblasts to mature vascular cells and our findings identified new mechanisms we can explore to better reprogram cardiac fibroblasts. Additionally, FOXK1 is a known cell cycle regulator and together with this newly identified role in the cardiovascular system, it becomes an attractive molecule that could be used to promote cell cycle re-entry of adult cardiomyocytes following ischemic injury. Altogether these studies provide exciting data for the field of cardiac regeneration but future studies will be needed in vivo to determine the potential benefit of these molecules following cardiac injury.