Browsing by Author "Hilton, Isaac"

Now showing 1 - 4 of 4
  • Thumbnail Image
    ItemEmbargo
    Engineering chromatin modifiers as biological discovery tools and epigenome editors
    (2024-04-19) Goell, Jacob; Hilton, Isaac
    Epigenetics is the study of heritable traits driven by alterations in the genome that occur on top of the DNA sequences encoding our genome. These features include histone and DNA modifications, chromatin architecture, and transcription factors that enable the emergence of cell types and a diversity of traits that are responsive to external stimuli. The study of the epigenome has thus been important for understanding basic biology and human disease. Until the emergence of CRISPR/Cas systems for programmable gene/epigenome editing, the field of epigenetics relied heavily on observational studies and genome-wide correlations to reach conclusions on the role epigenetic features play. The rapid adoption of CRISPR-based tools allowed for the targeted modification of DNA and deposition of precise epigenetic modifications through the fusion of chromatin-modifying factors to a catalytically inactivated version of a CRISPR system. While promising in its use for both basic and translational applications, the field still requires improved tools and a better understanding of their function to address inherent toxicity and off-targeting concerns, especially with respect to histone acylation, a modification linked to gene activation. Using the widely adopted epigenome editing effector domain and histone acyltransferase, p300, I address these concerns by (1) engineering mutations into p300 to alter its acylation deposition profile and cytotoxicity and (2) applying multi-omics and functional genomics methods to characterize these effectors for off-targets and benchmark their utility in a non-coding enhancer mapping screen.
  • Thumbnail Image
    ItemEmbargo
    Optimization of CRISPR/Cas-based Programmable DNA Methylation
    (2024-04-05) Guerra Resendez, Rosa Selenia; Hilton, Isaac; Bashor, Caleb
    CRISPR/Cas-based epigenome editing systems have transformed biomedical research by providing transcriptional and epigenetic control of healthy and disease states. Even though synthetic biology tools for DNA methylation writing and erasing have been employed in different architectural configurations and recruitment strategies, efforts to improve their functionality have been limited. Thus, engineering approaches are underexplored and needed to enhance the enzymatic activity of these tools, in particular for therapeutic applications in primary cells and in vivo models. In this doctoral dissertation, I present the optimization and application of programmable DNA methylation in human cells using CRISPR/Cas9-based epigenome editing. In the first chapter, optimization of lentiviral delivery of CRISPR/Cas9-based epigenome editing tools in human cells was conducted to better understand the parameters that lead to efficient delivery of epigenome editing tools. In the second chapter, rational engineering of the programmable synthetic DNA methylation writer (dCas9-DNMT3A/3L) was used to characterize the differential DNA methyltransferase activity of each designed variant, which catalyzed a distinctive methylation deposition profile and magnitude which correlated to gene silencing in different human cell lines. In the third chapter, engineered variants of dCas9-DNMT3A/3L were delivered to human primary T cells in an attempt to model immune exhaustion by targeting the BATF3 gene. Transcriptional silencing of BATF3 led to reduced cytokine production in human primary T cells. However, further experiments are needed to confirm that the reduced gene expression is caused by the DNA methylation deposited by the dCas9-based epigenetic effectors or if other factors independent of this epigenetic mark might be affecting transcription at this locus. Overall, the use of rationally engineered variants of the dCas9-DNMT3A/3L present an opportunity to interrogate the mechanistic parameters involved in programmable DNA methylation deposition and enable tunable transcriptional repression in human cells.
  • Thumbnail Image
    Item
    Temporal analysis suggests a reciprocal relationship between 3D chromatin structure and transcription
    (Elsevier, 2022) Reed, Kathleen S. M.; Davis, Eric S.; Bond, Marielle L.; Cabrera, Alan; Thulson, Eliza; Quiroga, Ivana Yoseli; Cassel, Shannon; Woolery, Kamisha T.; Hilton, Isaac; Won, Hyejung; Love, Michael I.; Phanstiel, Douglas H.
    To infer potential causal relationships between 3D chromatin structure, enhancers, and gene transcription, we mapped each feature in a genome-wide fashion across eight narrowly spaced time points of macrophage activation. Enhancers and genes connected by loops exhibit stronger correlations between histone H3K27 acetylation and expression than can be explained by genomic distance or physical proximity alone. At these looped enhancer-promoter pairs, changes in acetylation at distal enhancers precede changes in gene expression. Changes in gene expression exhibit a directional bias at differential loop anchors; gained loops are associated with increased expression of genes oriented away from the center of the loop, and lost loops are often accompanied by high levels of transcription within the loop boundaries themselves. These results are consistent with a reciprocal relationship where loops can facilitate increased transcription by connecting promoters to distal enhancers, whereas high levels of transcription can impede loop formation.
  • Thumbnail Image
    ItemEmbargo
    The design, characterization, and application of CRISPR-based tool in human cells
    (2023-07-20) Cabrera, Alan Cavido; Hilton, Isaac
    Many human diseases are associated with dysregulated gene expression. A subset of these diseases arise from deviated functional protein expression from homeostatic regimes. Either not enough, or too much, functional protein expression can cause toxic accumulation of metabolites, dysregulate regulatory networks, or myriad other systems necessary to maintain homeostasis. The field of epigenetics was born with the hypothesis that some form of regulation above the genetic level could explain the diverse phenotypic traits observed within a multicellular organism possessing a single genotype. Now we understand that homeostasis arises when the rich interplay of epigenetic mechanisms functions correctly. With our ever growing set of CRISPR-based tools, we are on the precipice of targeted correction of aberrant gene expression. Although many of these tools have been designed, there are still limitations regarding the effectiveness of many gold standard tools with therapeutically relevant human cell types. Work in this thesis was completed to: (1) explore the similarities between natural and synthetic epigenetic engineering as well as understanding the importance and complexities that dictate the transcriptional responses associated with dCas9-based histone acetylation. (2) construct a modular user-defined platform for the precise recruitment of proteins with diverse applications in targeted transcriptional activation, mammalian metabolic engineering, and identifying synergistic interactions of endogenous proteins in situ.