Sumoylation activates Pax‐6, an important transcription factor for eye and brain development

Abstract

Pax‐6 is a master gene controlling eye and brain development. Four isoforms of Pax‐6, p46, p48, p43 and p32/33, have been detected in the vertebrate eye. Absence of the paired domain in the 32kDa Pax‐6 distinguishes it from the other three forms. How is the 32 kDa Pax‐6 acting as a transcription factor is unknown. In the present study, we demonstrated that activation of the 32 kDa Pax‐6 requires sumoylation. First, nuclear and cytoplasmic distributions of Pax‐6 were detected in human lens epithelial cell line (FHL‐124) by Western‐blot analysis. Three Pax‐6 isoforms (46, 43 and 32 kDa) were detected in the nuclear extracts but only one isoform (43 kDa) was found in the cytoplasm. Second, a P3 sequence specific for HD domain was used to test the binding activities of different forms of Pax‐6 in gel mobility shifting assays. Only the 43kDa Pax‐6 displayed binding ability to the P3 sequence. 32 and 46 kDa Pax‐6 either extracted from FHL‐124 or synthesized in vitro lack the binding ability to P3 sequence. However, after incubating with the Pax‐6‐depeleted nuclear extract, 32kDa but not the 46kDa Pax‐6 exhibits strong binding ability to P3 sequence. Finally, both in vitro and in vivo biochemical analysis revealed the 32kDa Pax‐6 is sumoylated before binding to P3 sequence. Take together, the 32kda and 46kda Pax‐6 display differential DNA binding activity to the P3 sequence and may have differential regulatory mechanism on downstream target genes. Moreover, sumoylation of the 32 kda Pax‐6 is necessary to activate its transcriptional activity.