The Role of Protein-Protein Interactions in Controlling Prox1 Function

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University of Delaware
Prox1 is a homeodomain transcription factor that is important for controlling lens, liver, pancreatic, and lymphatic system development. Prox1 has also been implicated in the regulation of tumor suppressor genes and the onset of cancer. Despite this wide variety of functions, it is currently unknown as to how Prox1 is modulated during its role in vertebrate development. Previously, Arid2 (also called BAF200 and zipzap) was isolated from a yeast-two-hybrid assay as a potential Prox1 interacting protein. Arid2 belongs to the ARID family of proteins that are important for cell development, gene expression, and cell growth regulation. It is also a vital component of SWI/SNF complexes that function in chromatin remodeling. This work seeks to test the hypothesis that Prox1 and Arid2 actually interact and to determine the biological relevance of this interaction. First, a GAL4 yeast two-hybrid assay was used to confirm the protein interaction by the activation of reporter genes that are transcribed if the two proteins are able to bind. The plasmids Prox1-pGBKT7 and Arid2-pACT2, the yeast two-hybrid shuttle vectors, were transformed into two different yeast strains of two different mating types. These were mated and plated on selectable media lacking nutrients that the reporter genes are responsible for making. Yeast colonies did grow on selectable media, suggesting that Prox1 and Arid2 do interact. Furthermore, fluorescent immunohistochemical analysis was performed on four-week C57BL/6HAR mouse lens, which showed that Arid2 and Prox1 colocalize in the mouse lens fiber cells. After preliminary transfection analyses using the CAT reporter gene, it has been shown that Arid2 does not significantly affect Prox1- mediated activation of the βB1-crystallin promoter. These findings suggest that although Arid2 is able to bind Prox1, the functional consequences of this interaction may be difficult to decipher. Further research on other Prox1-interacting proteins may serve to elucidate the regulation of Prox1 in development.