Biochemistry of Prox1 function

Prox1 is a multifunctional transcription factor crucial for lens, lymphatic vessel, liver, pancreas, retina and brain development. It shares a highly conserved C-terminal domain containing the DNA-binding atypical homeodomain and a novel Prospero domain with its Drosophila homolog Prospero. Prox1 can work as either a transcriptional activator or a transcriptional co-repressor depending on the cellular and developmental environment. The goal of my work was to elucidate how Prox1 function is modulated through DNA binding and protein-protein interactions, mostly using the lens as a model.; I first produced a recombinant Prox1-GST fusion protein and used this as an antigen to create anti-Prox1 polyclonal and monoclonal antibodies. The polyclonal antibody produced in rabbit specifically detected Prox1 protein in lens tissue and lens-derived cell lines. 4G10 and 5G10, the two highly specific Prox1 monoclonal antibodies produced in mouse, have been licensed by several companies for use as a highly specific marker for the lymphatic vessels and the liver in cancer and metastasis research.; Prox1 can drive high level chicken betaB1-crystallin expression in the lens fiber cells. In addition to the identified Prox1-responsive OL2 element, I have characterized two more Prox1 binding elements, -220 and -290 elements in chicken betaB1-crystallin promoter, identified through DNAase I foot-printing. The -220 element was found to be a Prox1 repressive element with the highest binding affinity for Prox1 compared with the other two binding elements. The -290 element, which binds Prox1 less avidly, appeared to confer Prox1 repression as well. The results indicated that Prox1 can function as either a transcriptional activator or a repressor depending on the promoter context.; To identify proteins that might affect Prox1 function through protein-protein interactions, I created a Prox1 prey vector containing the conserved C-terminal homeo-Prospero domain and used the yeast two-hybrid assay to screen an 11.5 dpc mouse embryo cDNA library. The results indicated that Prox1 might interact with various proteins including cell cycle related proteins. Considering the dual roles of Prox1 in cell cycle regulation and its possible role as a tumor suppressor gene, I chose to further characterize Prox1 interactions with PCNA and PA2G4, two cell cycle related proteins.; Using yeast two-hybrid assays, I confirmed that Prox1 interacted with PCNA through the PCNA interacting protein motif (PIP box), located in the Prospero domain of Prox1, and mutation of the PIP box diminished the interaction. However, the integrated homeo-Prospero domain was required for the interaction, consistent with the X-ray structure demonstrating that these two domains constitute a single structure unit. Prox1 interacts with both the C-terminal domain and the IDCL (interdomain connecting loop) of PCNA. Both interactions were mediated through the PIP box as mutation of the PIP box diminished both interactions. In addition to the direct binding to the chicken betaB1-crystallin promoter, I confirmed that Prox1 directly bound to the Cyclin E promoter in gel shift assays. PCNA repressed Prox1 activation of both the betaB1-crystallin and the Cyclin E promoter in transfection assays, indicating that PCNA negatively regulates Prox1 function.; I also confirmed that PA2G4 interacted with the integrated homeo-Prospero domain. PA2G4 co-localized with Prox1 throughout lens development in immunofluorescence assays. PA2G4 significantly repressed Prox1 transcriptional activation of the OL2 element of chicken betaB1-crystallin promoter and of the Cyclin E promoter in transfection assays. The repression appears to be due to the ability of PA2G4 to impair Prox1 interactions with its binding sites since PA2G4 blocked Prox1 binding not only to the activating OL2 and Cyclin E elements, but also to the Prox1 repressive -220 and -290 elements in gel shift assays. These data highly suggest that Prox1 functions in c...