The process of reversible growth arrest, otherwise known as cellular quiescence or the G₀ phase denoted by withdrawal from the cell cycle, is a poorly characterized state. Subsets of growth arrest-specific (GAS) genes are upregulated during quiescence, however, these subsets are specific to/dependent on the limiting factor or circumstance inducing growth arrest. Here I characterize the expression and regulation of the lipid trafficking GAS gene Fatty Acid-Binding Protein 4 in the quiescence-inducing conditions of contact inhibition and oxygen limitation (hypoxia). Chicken Embryo Fibroblasts (CEF) were cultured to high density or subjected to hypoxia, in which oxygen is the limiting factor inducing growth arrest, or serum starvation, in which nutrients is the limiting factor inducing growth arrest. Contact inhibition and hypoxia induced FABP4 expression, whereas cycling control CEF and serum depleted CEF did not. At higher, though still hypoxic, oxygen levels that did not robustly induce FABP4, proliferation assays showed a slight reduction in CEF proliferation. The GAS gene p20k lipocalin has been shown to exhibit similar expression patterns to FABP4, with its regulation determined by the presence of the transcription factor C/EBP-β. CEF overexpressing C/EBP-β also showed strong FABP4 induction. Furthermore, chromatin immunoprecipitation (ChIP) assays revealed that C/EBP-β bound directly to the FABP4 promoter in both normoxic and hypoxic cells, although only the latter condition induced FABP4 protein expression. In summary, these results suggest that FABP4 is induced during growth arrest specifically when oxygen is the limiting factor, as induction was not seen during growth arrest mediated by starvation-induced endoplasmic reticulum (ER) stress, where nutrients was the limiting factor. The induction of these hypoxia-responsive genes suggests that oxygen availability regulates the expression of a sub-class of growth arrest specific genes. Additionally, FABP4 was shown to be associated with growth arrest and the promotion of cell survival and proliferation, as depicted by proliferation assays. Lastly, C/EBP-β not only strongly induced FABP4 expression, but directly bound to the FABP4 promoter. This suggests that C/EBP-β is a regulator of FABP4, although there may be other interacting factors acting as activators or repressors as this FABP4-C/EBP-β interaction was observed in conditions permissive and non-permissive to FABP4 expression. / Thesis / Master of Science (MSc) / The process of reversible growth arrest is a poorly characterized state. Subsets of growth arrest-specific (GAS) genes are upregulated during quiescence, however, these subsets are specific to the limiting factor or circumstance inducing growth arrest. Here we characterize the expression and regulation of the lipid trafficking GAS gene Fatty Acid-Binding Protein 4 in the quiescence-inducing conditions of contact inhibition (CI) and hypoxia. Chicken Embryo Fibroblasts (CEF) were cultured to high density or subjected to hypoxia, in which oxygen is the limiting factor inducing growth arrest, or serum starvation, in which nutrients availability is the limiting factor. CI and hypoxia induced FABP4 expression, whereas control and serum depleted CEF did not. At higher, though still hypoxic, oxygen levels that did not robustly induce FABP4, proliferation assays showed a slight reduction in CEF proliferation. When overexpressing C/EBP-β, CEF showed strong FABP4 induction. Additionally, a direct interaction with the FABP4 promoter was observed in both normoxic and hypoxic cells, although only the latter condition induced expression. In summary, the induction of this hypoxia-responsive gene suggests that oxygen availability regulates the expression of a sub-class of growth arrest specific genes and that this induction may be regulated by C/EBP-β.
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/24167 |
| Date | January 2018 |
| Creators | Peragine, Stephanie |
| Contributors | Bedard, Andre, Biology |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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