Pax-6 interactions with TATA-box-binding protein and retinoblastoma protein

PURPOSE. TO identify proteins that physically interact with Pax-G, a paired domain- and homeodomain (HD)-containing transcription factor that is a key regulator of eye development. METHODS. Protein-protein interactions involving Pax-6, TATA-box-binding pro tein (TPB), and retinoblastoma protein were studied using affinity chromato graphy with Pax-G as Ligand, glutathione-S-transferase (GST) pull-down assa ys, and immunoprecipitations. RESULTS. The authors have shown that Pax-G is a sequence-specific activator of many crystallin genes, all containing a TATA box, in the lens. Others h ave shown that lens fiber cell differentiation, characterized by temporally and spatially regulated crystallin gene expression, depends on retinoblast oma protein. In the present study it was shown that Pax-6 interacted with t he TBP, the DNA-binding subunit of general transcription complex TFIID. GST pull-down assays indicated that this interaction was mediated by the Pax-G HD, with a substantial role for its N-terminal arm and first two alpha-hel ices. The experiments also indicated a binding role for the C-terminal-acti vation domain of the protein. In addition, the present study showed that th e HD of Pax-G interacted with retinoblastoma protein. Immunoprecipitation e xperiments confirmed retinoblastoma protein/Pax-6 complexes in lens nuclear extracts. CONCLUSIONS. Blending the present results with those in the literature sugg ests that Pax-6 and retinoblastoma protein participate in overlapping regul atory pathways controlling epithelial cell division, fiber cell elongation, and crystallin gene expression during lens development.