Pax6 and SOX2 form a co-DNA-binding partner complex that regulates initiation of lens development

Pax6 is a key transcription factor in eye development, particularly in lens development, but its molecular action has not been clarified. We demonstrate that Pax6 initiates lens development by forming a molecular complex with SOX2 on the lens-specific enhancer elements, e.g., the delta-crystallin minimal enhancer DC5. DC5 shows a limited similarity to the binding consensus sequence of Pax6 and is bound poorly by Pax6 alone. However, Pax6 binds cooperatively with SOX2 to the DC5 sequence, resulting in formation of a high-mobility form of ternary complex in vitro, which correlates with the enhancer activation in vivo. We observed Pax6 and SOX2-interdependent factor occupancy of DC5 in a chromatin environment in vivo, providing the molecular basis of synergistic activation by Pax6 and SOX2. Subtle alterations of the Pax6-binding-site sequence of DC5 or of the inter-binding-sites distance diminished the cooperative binding and caused formation of a non-functional low-mobility form complex, suggesting DNA sequence-guided and protein interaction-induced conformation change of the Pax6 protein. When ectopically expressed in embryo ectoderm, Pax6 and SOX2 in combination activate delta-crystallin gene and elicit lens placode development, indicating that the complex of Pax6 and SOX2 formed on specific DNA sequences is the genetic switch for initiation of lens differentiation.