Identification of CUL9-related signaling pathways in human pluripotent stem cells and cortical precursors
Ortolano, Natalya Anne
The body of work presented in this dissertation details the characterization of a novel human-cell derived model of the understudied, non-canonical cullin scaffold CUL9. Unlike other cullins, previous efforts to characterize CUL9 function during the early stages of development using mouse KO models have been unsuccessful (Gama et al., 2014; Pei et al., 2011; Skaar et al., 2005, 2007). In Chapter 2, the development of a CUL9 KO hiPSC cell line using CRISPR-Cas9 is outlined. The line has normal expression of key pluripotent markers and is capable of neuronal differentiation, though there are abnormalities. With the advent of this new model, we sought to identify new CUL9 interacting proteins including substrates, regulators, and adapters that could provide insight into the aberrant neuronal differentiation observed through the experiments described in Chapter 3 and Chapter 4. The application of mass spectrometry based proteomic approaches to characterize CUL9 have also been unsuccessful in the past by our group and others (Gama et al., 2014; Li et al., 2014), however, we were able to successfully identify the APC/C subunit APC7 as a possible CUL9 regulating protein through LC-MS/MS analysis of immunoprecipitation of endogenous CUL9 protein in hiPSCs – this finding is detailed in Chapter 3. However, we were unable to fully elucidate the function or biochemical properties of this interaction leaving the question of CUL9’s function during neuronal differentiation unanswered. We attempted to remedy this in the study described in Chapter 4 though quantitative analysis of the proteome in CUL9 KO hiPSCs and differentiated neural precursors to identify proteins differentially expressed in KO cells compared to WT cells. We identified proteins involved in fatty acid metabolism and neuronal transcription but were unable to confirm their functional interaction with CUL9. However, we identified over 100 proteins differentially expressed in CUL9 KO cells which can be studied in the future to analyze the dynamic function of CUL9 during neuronal differentiation. In Chapter 5, I critically discuss the data presented and provide insight into the potential function of CUL9 in neuronal differentiation and how future studies can finally reveal the function of this mysterious and elusive enzyme.