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X. Chen, M.K. Duncan; Transcriptional Potential Of Prox1 . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2563.
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Prox1 is a transcription factor which has a highly conserved C–terminus containing a divergent homeodomain and novel Prospero domain. This work was conducted to study the function of the homeo–Prospero domain and how this function is modulated by interactions of this domain with other proteins.
The transcriptional activation domain of Prox1 was mapped using a modified mammalian one hybrid approach. Human Prox1 and a series of deletion fragments were fused to the Gal4 DNA binding domain in the eukaryotic vector pM. CHO cells were transfected with these constructs together with a reporter vector containing the reporter gene downstream of five consensus Gal4–responsive elements. A yeast two hybrid screen of a 11 day embryo mouse cDNA library was performed using the Prox1 homeo–Prospero domain as the bait protein. The expression pattern of the candidate Prox1 interacting proteins in the lens was determined by RT–PCR, western blot and confocal immunofluorescence. The influence of these proteins on the transcriptional potential of Prox1 was determined by co–transfection analysis.
In mammalian one hybrid assay, the activation activity of the Prox1 homeo–Prospero domain was 18 fold greater than the basal level. However, the homeodomain construct was only two fold over basal level whereas the Prospero domain alone conferred an 8 fold increase in activity. From the two–hybrid screen, we obtained diverse proteins that may interact with Prox1 in vivo. Of these proteins, Proliferation–associated 2G4 (PA2G4), a protein with established roles in cell cycle control, was found co–expressed with Prox1 during lens development. In transfection assays, we used a reporter consisting three consensus Prox1–responsive PL2 elements derived from the chicken ßB1–crystallin promoter placed upstream of the CAT reporter gene driven by the ßActin basal promoter. Prox1 activates this reporter whereas PA2G4 itself had no effect. When PA2G4 and Prox1 were co–transfected, PA2G4 significantly repressed Prox1 activation.
The highly conserved homeo–Prospero domain of Prox1 can function as a transcriptional activation domain while neither the homeo– or Prospero–domain alone works as efficiently. The results suggest that the activation activity of Prox1 needs the integrity of the homeo–Prospero 3–D structure, which corresponds to the X–ray result that these two domains constitute a single structural unit. The cell cycle regulator PA2G4 interacts with the homeo–Prospero domain and interferes with the ability of Prox1 to activate transcription at the native Prox1 binding site. In all, our work will help elucidate how Prox1 controls fiber cell differentiation and elongation in lens development.
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