Growth arrest specific (GAS) genes are highly inducible at quiescence (G0) and repressed rapidly in response to mitogens. Aberrant disruption of quiescence can lead to abnormal development and diseases such as cancer, thus, it is important to study the signals and mechanisms responsible for expressions of quiescent specific genes. p20K, a GAS gene whose expression is highly induced in conditions of contact inhibition & hypoxia in chicken embryo fibroblasts (CEF), is studied in this thesis. Preliminary studies demonstrate that p20K activation is dependent on its Quiescence Responsive Unit (QRU), a 48bp promoter region. In addition, the binding sites of a CCAAT/enhancer binding protein (C/EBPβ) and ERK2 on the QRU of p20K promoter overlap with each other regulating the competition between activating (C/EBPβ) and inhibiting (ERK2) of the p20K gene. After culturing CEF with media rich in growth factors (10%FBS), p20K induction is delayed in hypoxia. Moreover, it is the decrease of Phospho-ERK not CHOP level that correlates with p20K inhibition in hypoxia in both 5%CCS and 10%FBS. Western blotting analysis of Hypoxia Inducible Factor 1α (HIF1α) expression indicated that this hypoxia-response factor is induced rapidly and with the same kinetics in CEF subjected to hypoxia cultured in 5%CCS or 10%FBS, indicating that they sense and respond similarly to low oxygen concentrations. These results suggest that p20K induction in hypoxia is caused by growth arrest induced by hypoxia. To further document this process, hypoxia mimicking reagent DMOG, a prolyl-hydroxylase inhibitor that can stabilize HIF in normoxia, was used. Interestingly, p20K expression was highly induced after DMOG treatment in CEF, even if CHOP, an inhibitor of C/EBPβ, was induced in these conditions. Co-Immunoprecipitation results showed that the accumulation of CHOP-C/EBPβ heterodimers was induced during DMOG treatment. Additionally, Proliferation Assay suggested that DMOG treatment significantly inhibited CEF proliferation. Finally, Chromatin Immunoprecipitation Analysis indicated that ERK-2 did not bind to the QRU after DMOG treatment, indicating that ERK-2 dissociation correlates with p20K induction in response to DMOG in CEF. Collectively, these results demonstrate that growth arrest induced by hypoxia or DMOG treatment plays a determinant role in p20K induction. In contrast, CHOP level or CHOP-C/EBPβ heterodimer reduction did not correlate with the induction of p20K. / Thesis / Master of Science (MSc)

<p>A microarray analysis of RNA from contact inhibited CEF indicated a hypoxic signature in the contact inhibition program of gene expression (Ghosh <em>et al</em>., 2009). The purpose of this thesis was to investigate whether GAS genes known to be induced during contact inhibition are inducible by hypoxia. The gene p20K was selected as the model for this investigation because it is a growth arrest specific (GAS) gene with a well-characterized promoter (Mao <em>et al</em>., 1993). p20K expression was shown to be positively regulated in hypoxia. It was then determined by transient expression assay that this induction occurred at the promoter level. Interestingly by dissecting the promoter it was found that the quiescent responsive unit (QRU) was required for promoter induction during hypoxia. It has previously been shown that the QRU was required for contact inhibition induction of p20K in a C/EBPβ dependent manner (Mao <em>et al</em>., 1993; Kim <em>et al</em>., 1999).</p> <p>The mechanism behind hypoxic induction of the QRU was then investigated. The kinetics of HIF1α and p20K induction during hypoxia demonstrated that HIF1α was transiently expressed between 2-8 hrs of hypoxia while p20K was induced after 8 hrs of hypoxia. Co-Immuniprecipitation assay was also used to determine if a HIF1α-C/EBPβ interaction occurred, however, this molecular interaction could not be shown. These experiments suggests that HIF1α is not involved with the induction of the QRU. Over-expression of the dominant negative C/EBPβΔ184 repressed p20K induction, thus implicating C/EBPβ in activation in both contact inhibition and hypoxia. We also observed by western blot analysis that the C/EBP family member CHOP was repressed during hypoxia, causing a decrease in the amount of CHOP-C/EBPβ complexes in the cell. It was also found that over-expression of CHOP antagonized the induction of p20K by hypoxia. In conclusion hypoxia represses CHOP levels resulting in an increase of potent C/EBPβ homodimers at the expense of the inactive CHOP-C/EBPβ heterodimers.</p> / Master of Science (MSc)

<p>The study of growth arrest specific (GAS) genes is critical for our understanding of quiescence cell states. C/EBP-β is a transcriptional activator which is central to the expression of GAS genes in growth arrested cells. C/EBP-β is involved in the activation of numerous pathways, including mitogenesis, cytokine signaling, stress response, etc. Thus, it requires signaling cues which confer specificity in terms of gene expression.</p> <p>Here we used the p20K gene in chicken embryonic fibroblasts as a model system to study the control mechanisms of GAS genes. p20K is expressed in conditions such as contact inhibition mediated growth arrest and mild hypoxia. Here we explored the control mechanism mediated by ERK2 at the p20K promoter (QRU), as a mode of regulation which confers C/EBP-β binding specificity.</p> <p>In this study we demonstrate that ERK2 is recruited to the QRU in proliferative cells, i.e. where p20K is repressed. Using ChIP analysis we show that ERK2 binds directly to the QRU in proliferative cell states, but not in growth arrested cell conditions. Using a similar approach we demonstrate that ERK2 binding to the QRU is lost in states of hypoxia, where p20K is strongly induced. Furthermore, we show that this interaction is specific to ERK2 and is not observed with the related ERK1 kinase. Lastly, we employed transient expression assays to illustrate that ERK2 acts as a transcriptional repressor of the QRU. Through these experiments we have illustrated that ERK2 mediated transcriptional repression is a novel control mechanism at the QRU which skews C/EBP-β mediated signaling networks in proliferating cells.</p> / Master of Science (MSc)

Conditions which promote reversible growth arrest, such as hypoxia and high cell density, lead to activation of a diverse network of proteins known as growth arrest specific (GAS) genes. Fatty acid binding protein 4 (FABP4), a lipid chaperone involved in the regulation of metabolic and inflammatory responses, has been shown to be part of the GAS program. While the induction of FABP4 in oxygen-deprived environments is well characterized, its functionality and regulation in such conditions remains unclear. In this study, we describe how mis-expression of FABP4 affects cell viability and survival within low oxygen conditions. Loss of FABP4 using shRNA was shown to be associated with a significant increase in oxidative stress and lipid peroxidation, a reduction in lipid droplet formation and a greater incidence of apoptosis. Hypoxia-mediated expression of FABP4 was also found to be positively correlated with cellular levels of C/EBP-beta, an essential activator of p20K in quiescence. FABP4 and p20K are both lipocalins that have been shown to share similar induction patterns and ability to assist in the maintenance of lipid trafficking in cellular stress circumstances. Unexpectedly, the depletion of FABP4 or p20K results in loss of the other in limited oxygen concentrations. This occurs independently of disruption to the broad GAS gene program, suggesting the two proteins may be co-regulated in a shared hypoxic-signalling pathway. C/EBP-beta appears to be the transcriptional activator shared by FABP4 and p20K in quiescence, and the three may be part of an intricate system to sense and respond to reactive oxygen species and lipid radicals. However, the forced expression of either FABP4 or p20K when the other is repressed only moderately restores cell survival through alleviating oxidative stress, indicating the two are both necessary for optimal response to hypoxia. In all, these studies suggest that analogous to the p20K lipocalin, FABP4 plays a critical role in lipid homeostasis and cell survival in conditions of limited oxygen concentrations, and its stimulation is dependent on C/EBP-beta activity. / Thesis / Master of Science (MSc) / A study investigating the role of FABP4 and p20K in conditions of reversible growth arrest with an emphasis on cell survival, lipid homeostasis and mitigating the effects of oxidative stress, and regulation of the two lipocalins by C/EBP-beta.