Cell-type-specific epigenetics of organ-derived neurons and adipocytes
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Epigenetics has been the study of cell-type-specific differences in chromatin structure(s) (defined herein as epitype) among cells with the same genotype. The “Holy Grail” of current epigenetic is the cell-type-specific analyses of epitype within a complex tissue. Yet most of the publications on epigenetics examine the epitype of whole organs or parts of organs, which are comprised of dozens if not hundreds of cell types. In this dissertation, two technologies are developed, the first inspects the nuclei of specific cell types, Fluorescence Nuclear Cytometry (FNC), while the second both inspects and enriches the nuclei of specific cell types for further analyses, Fluorescence-Activated Nuclear Sorting (FANS). Two different mammalian organs were examined. FANS and FNC were employed to study subsets of neuronal nuclei from mouse brain and subsets of adipocyte nuclei from pig adipose depots. In all studies of this dissertation, cell-type-specific differences were observed in those subpopulations fractionated by FANS. The first study (Chapter 3) determined that a significant subpopulation of adult mouse brain nuclei (NeuN-High) had highly decondensed chromatin, expressed elevated levels of chromatin modifying machinery, and expressed high levels of transcripts encoding markers of neurogenesis, learning and memory, multipotency and cell cycle activity. The surprising discovery is the co-expression of multipotency and neuronal activity markers in the same subpopulation. In chapter 4, 5-hydroxymethylcytosine (5hmC), the first product in the demethylation of 5-methylcytosine (5mC), levels were evaluated among NeuN-High, NeuN-Low and NeuN-Neg mouse brain nuclei. Tet-assisted bisulfite sequencing (TAB-seq) demonstrated that 5hmC levels in NeuN-High nuclei were significantly higher compared to NeuN-low and NeuN-Neg nuclei. The third study (chapter 5) identified a previously unknown subset of visceral adipose tissue (VAT) nuclei (PPARg2-Positive) that expressed significantly higher levels of transcripts encoding both mature adipocyte markers and chromatin-remodeling factors, and also had higher levels of 5hmC. Collectively, the studies of this dissertation provide further evidence that epigenetic reprogramming is a cell-type-specific process and advance nuclear cytometry for the analysis of brain and adipose tissues. Future epigenetics analyses will be enhanced by using the full potential of FANS and FNC technologies along with the wide variety of new and well-characterized immunological markers.