Date of Award
Doctor of Philosophy (PhD)
Proper cellular development and function is a complex process established by elaborate gene expression networks. These networks are regulated by epigenetic processes, which alter chromatin states and coordinate the binding of transcription factors (TFs) to regulatory elements (REs), such as enhancers, across the genome to facilitate gene expression. It follows then that a major experimental effort is to profile and understand the binding patterns of TFs to REs in various cellular types and contexts. Critically however, current TF profiling techniques are limited in their abilities to profile TF occupancy in targeted cellular populations and temporal windows, hindering investigations into epigenetic control in complex, multicellular systems, such as the brain. This dissertation focuses on two related areas: firstly, the design of new tools for profiling TF genome occupancy in the mouse brain in specific cellular populations and time periods, and secondly, investigating TF-mediated mechanisms of disease pathogenesis in animal models. In Chapter 2, we describe the development of a novel, viral-mediated method, termed adeno-associated virus (AAV) calling cards, for profiling binding sites of TFs across the genome in the live mouse brain. The AAV calling cards approach allows unique access to TF occupancy information that is inaccessible with other existing techniques, including cell type specificity (through Cre-mediated conditional expression) and historical binding (through longitudinal occupancy recording). Then, in Chapters 3 and 4, we apply this new technique to mouse models to investigate epigenetic misregulation in disease. Previous studies have demonstrated that a large portion of genetic variation associated with cellular dysfunction or disease exists in TF-bound enhancers, demonstrating the criticality of proper TF binding in maintaining cellular homeostasis. However, whether these elements are misregulated more broadly in disease contexts is unclear. In Chapter 3, we apply AAV calling cards to a model of acute seizure and uncover aberrant epigenetic regulation which is predictive of phenotypic outcomes. Particularly important in this study is the ability of AAV calling cards to record and integrate historical TF binding information, allowing linkage of antecedent epigenetic events to eventual seizure outcomes. Here, we longitudinally recorded prodromal enhancer activity to identify loci which are predictive of seizure severity. Next, in Chapter 4, we investigate epigenetic regulation in animal models and postmortem tissues from individuals with amyotrophic lateral sclerosis (ALS). In this study, we focus on a subset of ALS caused by a large hexanucleotide (G4C2) repeat expansion in the gene chromosome 9 open reading frame 72 (C9orf72), which is the most common genetic cause of ALS (C9ALS). Utilizing AAV calling cards as well as other established epigenomic profiling techniques, we observe broad epigenetic misregulation both in C9ALS mouse models and human tissues at the transcriptional and translational levels. Importantly, the C9ALS mouse models used in this study do not develop motor neuron degeneration or ALS-like phenotypes and were profiled at an early age, suggesting that these changes occur early in the disease process and are likely driven by C9orf72-related pathologic species, such as dipeptide repeat proteins (DPRs). Finally, in Chapter 5 we investigate the characteristic properties of C9orf72-specfic pathologies, including DPRs, in human C9ALS. We probed size and abundance of DNA expansions and DPRs in blood, cerebrospinal fluid, and postmortem tissues from C9ALS and sporadic ALS (sALS) individuals and identified novel correlations of C9ALS patient pathologies with clinical and demographic data. Moving forward, these data will facilitate mechanistic studies and clinical trials aimed at reducing or altering C9ALS pathologies in the central nervous system (CNS). In summary, the body of work detailed here extends our knowledge of TFs in both the healthy and diseased central nervous system (CNS), providing new insights into the role of epigenetic regulation in disease pathogenesis. Further, the establishment of AAV calling cards as a widely applicable epigenomic tool will empower innovative new studies in a variety of tissue and model systems.
Chair and Committee
Timothy M. Miller
Joseph D. Dougherty, Harrison W. Gabel, Robi D. Mitra, Erik S. Musiek,
Cammack, Alexander J., "Transcription Factor-Mediated Epigenetic Regulation in the Healthy Brain and Neurological Disease" (2020). Arts & Sciences Electronic Theses and Dissertations. 2170.