"Investigating stem cell chromatin organization and oncogenic tissue mo" by Sangwon Yun

Date of Award

Fall 2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Genetics

First Advisor

Greco, Valentina

Abstract

Tissue architecture is predicated on actively maintained cellular equilibrium. Loss of homeostatic tissue organization is a hallmark of epithelial derived malignancy. In the following studies, we attempt to understand the principles governing cell behavior in a regenerative tissue, the skin epidermis, under homeostatic conditions and during oncogenic expression. Epidermal cells undergo continual proliferation and differentiation, navigating drastic changes in gene expression and chromatin architecture during identity transition from stem to differentiated. However, when and where genome reorganization occurs continues to be an arena of active investigation. We developed a high-throughput, quantitative pipeline that enabled us to longitudinally measure chromatin compaction in single cells in live mice. Stem and differentiated cells exhibited distinct chromatin compaction states, and chromatin compaction heterogeneity within the stem cell compartment reflected the evolution in cell identity as cells underwent progressive, directional shifts in chromatin compaction concordant with differentiation and exit from the stem cell compartment. By visualizing transcription of the early differentiation-associated gene keratin-10, we found that expression of this gene is highly dynamic and precedes chromatin architecture changes. Altogether, cell identity transition from stem to differentiated is characterized by progressive large-scale genome reorganization and dynamic transcriptional behavior. We also sought to understand how cancer-associated mutation expression provokes loss of homeostatic tissue architecture. Epidermal oncogenic β-catenin expression forms orthogonal axes of aberrant growth. However, the earliest cellular changes associated with and the molecular conditions required for growth remain to be elucidated. In particular, the ways in which the dual functions of β-catenin as a cortical junctional component and nuclear Wnt effector coordinate tissue morphological changes remain to be understood. As such, we sought to observe overtime tagged, oncogenic β-catenin in live mice, imaging inducible "β" -cateninΔ90-GFP in adult murine skin using two-photon microscopy. We first explored how cell shape and cell-cell adhesion evolves prior to disruption in tissue architecture. Measuring stem cell cross sectional area, we found mutant cells, then their wild-type neighbors, shrink prior to growth formation, suggesting local mechanical disruption incurs regional changes while wild-type—mutant cell interaction defines cell shape in the earliest stages of oncogenic expression. We then investigated the preconditions for growth. As stem cell nuclear "β" -catenin intensity correlated with aberrant growth formation, we wondered whether differentiating stem cells, roughly 40 percent of all stem cells, could also form growth. Interestingly, mutation expression in differentiating stem cells failed to form growth, yet they also had little to no nuclear "β" -catenin. As the loss of cell-cell adhesion provokes tumor progression and increases nuclear "β" -catenin by decreasing the cortical sink, we sought to rescue growth formation in differentiating stem cells by knocking down E-cadherin. Although nuclear "β" -catenin levels did indeed increase, no growths formed, suggesting coordination between molecular function and cell state are required to form oncogenic growth. Altogether, "β" -catenin seems to play multiple roles in cell morphology, adhesion and signaling during growth formation. In summary, our work on chromatin architecture, gene expression and oncogenic initiation offer exciting insight that may be leveraged toward better understanding and perhaps even clinically detecting the fine line between homeostasis and disease.

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