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Open Access Publications from the University of California

Scholarly Works (6 results)

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UC Irvine Electronic Theses and Dissertations (2022)

Phosphorylation dependent regulation of GLI transcription factors and Hedgehog signaling

By

Eric TaraporeDoctor of Philosophy in the Biological Sciences University of California, Irvine, 2022 Associate Professor Scott Atwood, Chair

Basal cell carcinoma (BCC) is one of the most prevalent cancers and is mainly driven by the overactivation of the Hedgehog (HH) signaling pathway. Although easily treatable via Smoothened inhibitor vismodegib, drug resistance is common and as such alternative avenues to treat BCCs must be identified. One way to target pathway activity is through targeting the transcription factors responsible for relaying the signals. Here we use a combination of molecular biology techniques to identify additional ways to regulate the HH signaling pathway. We identify phosphorylation as being a key regulator of GLI activity and binding. We identify that AURKA plays a key role in regulating BCC growth in vitro and in vivo. We found that pharmacological inhibition of AURKA in the Ptch1fl/fl; Gli1-CreERT2 mouse BCC tumor model resulted in significantly reduced levels of BCC cell growth. Inhibition of AURKA in BCC cells in vitro resulted in reduced levels of HH signaling and cell growth. This goes with work that was conducted showing that phospho-mimetic mutation of the zinc finger domain of GLI results in differential levels of DNA binding and activity. We identify a specific region of the zinc finger domain that is permissive to phospho-mimetic mutation whereas other regions of the zinc finger completely abolish DNA binding and activity. We have termed these as the permissive regulatory region along with the more restrictive DNA binding region and linker region. Further studies into additional zinc finger transcription factors seem to show similar results although not to the same extent as GLI. Together these finding identify alternate means of GLI regulation, either through phosphorylation of GLI via AURKA or phosphorylation of the zinc finger binding domain of GLI.

    Cover page: Phosphorylation dependent regulation of GLI transcription factors and Hedgehog signaling
    • Article
    • Peer Reviewed
    UC Irvine Previously Published Works (2022)
    Tissues and organs are composed of many diverse cell types, making cell-specific gene expression profiling a major challenge. Herein we report that endogenous enzymes, unique to a cell of interest, can be utilized to enable cell-specific metabolic labeling of RNA. We demonstrate that appropriately designed "caged" nucleosides can be rendered active by serving as a substrate for cancer-cell specific enzymes to enable RNA metabolic labeling, only in cancer cells. We envision that the ease and high stringency of our approach will enable expression analysis of tumor cells in complex environments.
      Cover page: Exploiting Endogenous Enzymes for Cancer-Cell Selective Metabolic Labeling of RNA in Vivo
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2018)
      Primary cilia are polarized organelles that allow detection of extracellular signals such as Hedgehog (Hh). How the cytoskeleton supporting the cilium generates and maintains a structure that finely tunes cellular response remains unclear. Here, we find that regulation of actin polymerization controls primary cilia and Hh signaling. Disrupting actin polymerization, or knockdown of N-WASp/Arp3, increases ciliation frequency, axoneme length, and Hh signaling. Cdc42, a potent actin regulator, recruits both atypical protein pinase C iota/lambda (aPKC) and Missing-in-Metastasis (MIM) to the basal body to maintain actin polymerization and restrict axoneme length. Transcriptome analysis implicates the Src pathway as a major aPKC effector. aPKC promotes whereas MIM antagonizes Src activity to maintain proper levels of primary cilia, actin polymerization, and Hh signaling. Hh pathway activation requires Smoothened-, Gli-, and Gli1-specific activation by aPKC. Surprisingly, longer axonemes can amplify Hh signaling, except when aPKC is disrupted, reinforcing the importance of the Cdc42-aPKC-Gli axis in actin-dependent regulation of primary cilia signaling.
        Cover page: Actin polymerization controls cilia-mediated signalingCreative Commons 'BY' version 4.0 license
        • Article
        • Peer Reviewed
        UC Irvine Previously Published Works (2019)

        ABSTRACT

        How stem cells give rise to human interfollicular epidermis is unclear despite the crucial role the epidermis plays in barrier and appendage formation. Here we use single cell-RNA sequencing to interrogate basal stem cell heterogeneity of human interfollicular epidermis and find at least four spatially distinct stem cell populations that decorate the top and bottom of rete ridge architecture and hold transitional positions between the basal and suprabasal epidermal layers. Cell-cell communication modeling through co-variance of cognate ligand-receptor pairs indicate that the basal cell populations distinctly serve as critical signaling hubs that maintain epidermal communication. Combining pseudotime, RNA velocity, and cellular entropy analyses point to a hierarchical differentiation lineage supporting multi-stem cell interfollicular epidermal homeostasis models and suggest the “transitional” basal stem cells are stable states essential for proper stratification. Finally, alterations in differentially expressed “transitional” basal stem cell genes result in severe thinning of human skin equivalents, validating their essential role in epidermal homeostasis and reinforcing the critical nature of basal stem cell heterogeneity.
          Cover page: Single cell transcriptomics of human epidermis reveals basal stem cell transition states
          • Article
          • Peer Reviewed
          UC Irvine Previously Published Works (2018)
          The genetically heterogeneous spinocerebellar ataxias (SCAs) are caused by Purkinje neuron dysfunction and degeneration, but their underlying pathological mechanisms remain elusive. The Src family of nonreceptor tyrosine kinases (SFK) are essential for nervous system homeostasis and are increasingly implicated in degenerative disease. Here we reveal that the SFK suppressor Missing-in-metastasis (MTSS1) is an ataxia locus that links multiple SCAs. MTSS1 loss results in increased SFK activity, reduced Purkinje neuron arborization, and low basal firing rates, followed by cell death. Surprisingly, mouse models for SCA1, SCA2, and SCA5 show elevated SFK activity, with SCA1 and SCA2 displaying dramatically reduced MTSS1 protein levels through reduced gene expression and protein translation, respectively. Treatment of each SCA model with a clinically approved Src inhibitor corrects Purkinje neuron basal firing and delays ataxia progression in MTSS1 mutants. Our results identify a common SCA therapeutic target and demonstrate a key role for MTSS1/SFK in Purkinje neuron survival and ataxia progression.
            Cover page: MTSS1/Src family kinase dysregulation underlies multiple inherited ataxias
            • Article
            • Peer Reviewed
            UC Irvine Previously Published Works (2020)
            How stem cells give rise to epidermis is unclear despite the crucial role the epidermis plays in barrier and appendage formation. Here we use single cell-RNA sequencing to interrogate basal stem cell heterogeneity of human interfollicular epidermis and find four spatially distinct stem cell populations at the top and bottom of rete ridges and transitional positions between the basal and suprabasal epidermal layers. Cell-cell communication modeling suggests that basal cell populations serve as crucial signaling hubs to maintain epidermal communication. Combining pseudotime, RNA velocity, and cellular entropy analyses point to a hierarchical differentiation lineage supporting multi-stem cell interfollicular epidermal homeostasis models and suggest that transitional basal stem cells are stable states essential for proper stratification. Finally, alterations in differentially expressed transitional basal stem cell genes result in severe thinning of human skin equivalents, validating their essential role in epidermal homeostasis and reinforcing the critical nature of basal stem cell heterogeneity.
              Cover page: Single cell transcriptomics of human epidermis identifies basal stem cell transition states
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