Regulation of Gene Expression in Albumin-alpha-fetoprotein Locus
University of Kentucky, 2008 - 106 pages
My first project was to study the role of Foxa2 proteins on AFP promoter activity. Foxa2 is a liver-enriched transcription factor important for liver development. My experiments demonstrated that Foxa2 and its DNA binding domain alone could repress AFP promoter activity in transiently transfected HepG2 cells. A region between -205 bp and -150 bp of AFP promoter conferred the Foxa2-mediated repression on a heterologous promoter in HepG2 cells, and this repression was lost when the -165 site in this region was mutated. Using a series of site-directed and truncated Foxa2 mutants, I showed that DNA binding activity of Foxa2 is not required for this repression, but nuclear localization is required. This indicates that Foxa2 repress AFP promoter through an indirect mechanism. Using Foxa2-DBD-GST affinity chromatography coupled to Mass Spectrometry analysis, several potential proteins that might interact with Foxa2 DNA binding domain were identified. Further studies will be needed to determine whether these Foxa2 bound proteins are involved in AFP repression in HepG2 cells.
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