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Open Access Publications from the University of California

Scholarly Works (4 results)

  • Thesis
  • Peer Reviewed
UC Davis Electronic Theses and Dissertations (2022)

Salinity in aquatic environments limits the abundance and distribution of fish in a particular ecological niche. Mozambique tilapia (Oreochromis mossambicus) can tolerate a wide range of salinity stress (they are euryhaline). Such stress is currently greatly intensified in many aquatic environments due to anthropogenically accelerated climate change. Effective osmoregulation is critical for fish and other aquatic organisms to adapt to salinity stress. Physiological and biochemical approaches to understand osmoregulatory mechanisms of fish have previously identified many genes, proteins, and biochemical pathways associated with compensatory responses to salinity stress. However, the underlying molecular mechanisms that control the osmotic regulation of these genes and pathways are still largely elusive. To address this knowledge gap, the overall objective of my thesis was to identify molecular underpinnings of cellular osmoregulatory mechanisms that are utilized by euryhaline fish. We used a cell line model (tilapia cell line) to study how osmoregulated genes are activated by hyperosmotic stress. The emphasis was on identifying and characterizing cis-elements and trans-factors that control osmotic regulation of gene expression by utilizing the advantages of this cell line as a genetically tractable experimental system. Salinity-responsive cis-regulatory elements (CREs) and their role in the hyperosmotic induction of the tilapia glutamine synthetase gene were identified and characterized using a targeted approach (Chapter 2). A systematic non-targeted approach that utilized bioinformatics and experimental tools was used to discover new osmoregulated CREs (Chapter 3). This non-targeted approach was based on enrichment of DNA sequence motifs in promoters of hyperosmotically upregulated genes. STREME1 was identified and experimentally validated as a new salinity-responsive CRE (Chapter 3). Lastly, CRISPR/Cas9 technology was used to engineer mono- and polyclonal tilapia cell lines that harbor a functionally inactive MYC transcription factor (TF) to enable future loss-of-function studies (Chapter 4). Specifically, using a modified limiting dilution strategy several polyclonal knockout (ko) cell lines (heterogeneous cell pools) and a monoclonal myca ko cell line were generated. Most of the evolutionarily conserved functional domains of MYC were removed in the monoclonal ko cell line. The knowledge and tools generated in this dissertation research advance our understanding of the molecular mechanisms that link changes in environmental salinity and extracellular osmolality with the transcriptional regulation of specific sets of genes that underlie compensatory responses to salinity stress.

    Cover page: Investigating transcriptional regulatory mechanisms of euryhaline fish in response to salinity stress using a tilapia cell line
    • Article
    • Peer Reviewed
    UC Davis Previously Published Works (2023)
    MYC transcription factors have critical roles in facilitating a variety of cellular functions that have been highly conserved among species during evolution. However, despite circumstantial evidence for an involvement of MYC in animal osmoregulation, mechanistic links between MYC function and osmoregulation are missing. Mozambique tilapia (Oreochromis mossambicus) represents an excellent model system to study these links because it is highly euryhaline and highly tolerant to osmotic (salinity) stress at both the whole organism and cellular levels of biological organization. Here, we utilize an O. mossambicus brain cell line and an optimized vector-based CRISPR/Cas9 system to functionally disrupt MYC in the tilapia genome and to establish causal links between MYC and cell functions, including cellular osmoregulation. A cell isolation and dilution strategy yielded polyclonal myca (a gene encoding MYC) knockout (ko) cell pools with low genetic variability and high gene editing efficiencies (as high as 98.2%). Subsequent isolation and dilution of cells from these pools produced a myca ko cell line harboring a 1-bp deletion that caused a frameshift mutation. This frameshift functionally inactivated the transcriptional regulatory and DNA-binding domains predicted by bioinformatics and structural analyses. Both the polyclonal and monoclonal myca ko cell lines were viable, propagated well in standard medium, and differed from wild-type cells in morphology. As such, they represent a new tool for causally linking myca to cellular osmoregulation and other cell functions.
      Cover page: Removal of evolutionarily conserved functional MYC domains in a tilapia cell line using a vector-based CRISPR/Cas9 system.
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2024)
      Variants in cis-regulatory elements link the noncoding genome to human pathology; however, detailed analytic tools for understanding the association between cell-level brain pathology and noncoding variants are lacking. CWAS-Plus, adapted from a Python package for category-wide association testing (CWAS), enhances noncoding variant analysis by integrating both whole-genome sequencing (WGS) and user-provided functional data. With simplified parameter settings and an efficient multiple testing correction method, CWAS-Plus conducts the CWAS workflow 50 times faster than CWAS, making it more accessible and user-friendly for researchers. Here, we used a single-nuclei assay for transposase-accessible chromatin with sequencing to facilitate CWAS-guided noncoding variant analysis at cell-type-specific enhancers and promoters. Examining autism spectrum disorder WGS data (n = 7280), CWAS-Plus identified noncoding de novo variant associations in transcription factor binding sites within conserved loci. Independently, in Alzheimers disease WGS data (n = 1087), CWAS-Plus detected rare noncoding variant associations in microglia-specific regulatory elements. These findings highlight CWAS-Pluss utility in genomic disorders and scalability for processing large-scale WGS data and in multiple-testing corrections. CWAS-Plus and its user manual are available at https://github.com/joonan-lab/cwas/ and https://cwas-plus.readthedocs.io/en/latest/, respectively.
        Cover page: CWAS-Plus: estimating category-wide association of rare noncoding variation from whole-genome sequencing data with cell-type-specific functional data.
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2018)
        Although abnormal increases in the level or activity of cyclin-dependent kinase 4 (CDK4) occur frequently in cancer, the underlying mechanism is not fully understood. Here, we show that methionyl-tRNA synthetase (MRS) specifically stabilizes CDK4 by enhancing the formation of the complex between CDK4 and a chaperone protein. Knockdown of MRS reduced the CDK4 level, resulting in G0/G1 cell cycle arrest. The effects of MRS on CDK4 stability were more prominent in the tumor suppressor p16INK4a-negative cancer cells because of the competitive relationship of the two proteins for binding to CDK4. Suppression of MRS reduced cell transformation and the tumorigenic ability of a p16INK4a-negative breast cancer cell line in vivo. Further, the MRS levels showed a positive correlation with those of CDK4 and the downstream signals at high frequency in p16INK4a-negative human breast cancer tissues. This work revealed an unexpected functional connection between the two enzymes involving protein synthesis and the cell cycle.
          Cover page: Stabilization of Cyclin-Dependent Kinase 4 by Methionyl-tRNA Synthetase in p16INK4a-Negative Cancer
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