Browsing by Author "Thompson, Elizabeth"

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    Identifying Precision Medicine for Dystrophic Epidermolysis Bullosa
    (2020-12) Thompson, Elizabeth
    Background: Dystrophic epidermolysis bullosa (DEB) is a skin blistering disease caused by mutations in the COL7A1 gene, which encodes type VII collagen (C7). There is potential therapeutic benefit for DEB patients in identifying compounds that can increase C7 production in skin. Methods: A keratinocyte C7 reporter cell line was created and used in a genome-wide CRISPR activation (CRISPRa) screen to identify genes and pathways that increase C7 expression. Based on the CRISPRa screen results, a targeted drug screen was performed in three different keratinocyte cell lines to evaluate C7 upregulation. Results and Conclusions: The C7_tdTomato cell line was validated as an effective C7 reporter cell line. The CRISPRa screen identified two genes, DENND4B and TYROBP as top hits and pathway analysis showed enrichment of immune signaling pathways and regulators. The targeted drug screen identified kaempferol as a potential precision medicine for DEB.
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    The Role of Fanconi Anemia Proteins in DNA Repair, Replication Stress and Genome Stability
    (2017-10) Thompson, Elizabeth
    Fanconi anemia (FA) is a genetic chromosomal instability disorder characterized by progressive bone marrow failure and a strong predisposition to cancer. The FA proteins work together in a cellular pathway for the repair of DNA interstrand crosslinks (ICLs). Currently 22 different FA genes are implicated in this disease and contribute to the heterogeneity in symptoms and severity. Using gene targeting techniques, we successfully created a set of isogenic knockout cell lines to represents all 3 groups of proteins within the FA pathway to characterize protein function and identify differences that help explain FA disease heterogeneity. In Chapter 2 we investigate the FA group 2 proteins, FANCI and FANCD2 that that form a heterodimer called the ID complex. We characterized the FANCI, FANCD2 and FANCI/FANCD2 double knockout cell lines and identified non-overlapping functions in the replication stress response. In fact, we found that only FANCD2 is required for restart of stalled replication forks and FANCI may even inhibit restart when FANCD2 is absent. In addition, FANCD2 has a more vital role in homologous recombination, and FANCI promotes apoptosis in the absence of FANCD2 with replication stress. In Chapter 3 we investigate FANCN, an FA group 3 protein that is associated with more severe FA disease. We used FANCN conditional knockout cells to determine that FANCN is essential for viability and genome stability. In addition, we evaluated FANCN FA-associated mutations and breast cancer-associated variants of unknown significance (VUS) mutations. We confirmed that the BRCA1/2 binding domains of FANCN are not essential for viability and identified two VUS mutations as potentially pathogenic. In conclusion, we have demonstrated alternative functions of FA proteins in response to replication stress as a potential source of FA disease heterogeneity. In addition, we have demonstrated that FANCN is essential for viability whereas FANCI and FANCD2 are not, providing insight into both the frequency of occurrence and severity of FA disease associated with these different genes. Finally, we created isogenic cell lines that are a valuable asset for the FA and breast cancer fields for further investigations into protein function, characterization of patient mutations, and screening novel therapeutics.