CHARACTERIZING THE ROLE OF CCAAT/ENHANCER BINDING PROTEIN β ON QUIESCENCE-SPECIFIC GENE EXPRESSION IN CHICKEN EMBRYO FIBROBLASTS

Abstract

<p>CCAAT/enhancer binding protein (C/EBP) family of transcription factors regulate cellular fates such as proliferation and differentiation. During conditions like serumdepletion or contact inhibition, cells can exit the cell cycle and enter a state of quiescence. Our studies have focused on characterizing the role of the C/EBP family in cellular survival and quiescence in chicken embryonic fibroblasts (CEF).</p> <p>Treating CEF with a dominant negative mutant of C/EBPβ (designated Δ184-C/EBPβ) increased cell survival during prolonged starvation. These cells also developed large autophagosomes characteristic of autophagy, a process cells utilize to acquire energy by degrading their own organelles. In yeast, a key mediator of autophagy is A TG8, which is essential for autophagosome formation and maintenance. Starved CEF expressing ΔI84-C/EBPβ had elevated levels of a vertebrate homologue of ATG8, Y(Gamma)<strong>-</strong>aminobutyric acid receptor-associated protein (GABARAP). Hence, we attempted to determine whether GABARAP plays a role in regulating cellular survival in starved CEF. However, over-expression and shRNAi studies indicate that GABARAP is not required for survival but instead may regulate apoptosis in CEF during starvation.</p> <p>We also attempted to determine the effects of the C/EBP family in quiescencespecific gene expression. p20K is a well-characterized quiescence-specific gene in CEF, whose expression is regulated by C/EBPβ and cellular density. Recent findings suggest that p20K expression is negatively regulated by another member of the C/EBP family, CHOP10. Gene-profiling studies also discerned that contact-inhibited CEF display a signature of hypoxia, where genes such as HIF1α(alpha) and carbonic anhydrase IX are activated. Further analyses show that hypoxia can activate p20K expression in a C/EBPβ-dependent manner and this induction is repressed by CHOP10. Additionally, HIF1α(alpha). may also be regulated by C/EBPβ, since its dominant negative mutant repressed HIF1α(alpha). expression in CEF treated with hypoxia mimetics. Therefore, these findings suggest the existence of a C/EBPβ-HIF1α(alpha). axis in the regulation of p20K expression in CEF.</p>