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Investigating transcriptional regulatory mechanisms of euryhaline fish in response to salinity stress using a tilapia cell line


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.

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