Date of Award
Master of Science (MS)
The interest in adipogenesis stems from the high rate of obesity in the world and the motivation to better understand the process of cell fate in relation to human diseases. Mutations in MED12 lead to developmental disorders and certain malignancies. To date, specific roles of Mediator and MED12 in cell state regulation are unclear, nevertheless, they are critical for understanding stem cell regulation and enhancement of their clinical applications. This study investigates the role of MED12 in relation to the Mediator complex kinase domain to better understand transcriptional control of adipogenesis in hASCs. In this study, MED12 expression was diminished with MED12 specific siRNA and adipogenesis was induced/assessed at different time points (day 3,7 and 14) using phase imaging to detect lipid droplets with Oil red-O staining being used to further validate differentiation and visualize the cells. To validate and confirm the effects of the MED12 knockdown, qRT-PCR and western blot analysis was carried out. To determine the role of MED12 as it functions in the Mediator complex and its role in maintaining the structural integrity of the kinase module, Coimmunoprecipitation assays were carried out. Results from this study show that in the absence of MED12, there is a significant decrease in the number of lipid vesicles compared to the negative control as observed via oil red-O staining and a decrease in the expression of adipogenic transcription factors such as PPARγ and C/EBPα via qRT-PCR and western blot analysis. Results also indicated that MED12 interacts with the adipogenic transcription factors to direct proper gene expression and indicates a role for MED12 and the Mediator complex in directing the process of transcription during adipogenesis. Taken together, these data suggest a significant role for MED12 in regulating adipogenesis and will initiate new research into understanding the regulatory mechanisms of adipogenesis and offer insight into novel treatments for obesity and relevant human disease.
Idigo, Onyekachi Chinelo, "" (2021). Thesis. 63.