Sumoylation of the 32 kD Pax-6 Activates Its Transcriptional Activity

Abstract

Purpose: : Pax-6 is a master regulator 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 32-kD Pax-6 distinguishes it from the other three forms. How is the 32-kD Pax-6 acting as a transcription factor remains unknown. In the present study, we demonstrated that activation of the 32-kD Pax-6 requires sumoylation, an important posttranslational modification.

Methods: : Western-blot analysis was used to detect the nuclear and cytoplasmic distributions of Pax-6 in human lens epithelial cell line (FHL-124). Gel mobility shifting assays were used to test the binding activities of different forms of Pax-6 to a P3 sequence specific for homeodomain. In vitro sumoylation assay was conducted to detect sumoylation of 32-kD Pax-6. Immunohistochemistry and co-immunoprecipitation were used to detect the in vivo interactions between Pax-6 and sumo proteins.

Results: : Three Pax-6 isoforms (46-, 43- and 32-kD) were detected in the nuclear extracts but only one isoform (43-kD) was found in the cytoplasm. Only the 43-kD Pax-6 displayed binding ability to the P3 sequence. The 32- and 46-kD Pax-6 synthesized in vitro lack the binding ability to P3 sequence. After incubating with the Pax-6-depeleted nuclear extract, the 32-kD but not the 46-kD Pax-6 exhibited strong binding ability to P3 sequence. This binding activity of 32-kD Pax-6 was prevented when the nuclear extract was pre-cleared with sumo antibodies. The interacting complexes of Pax-6 and sumo proteins were detected in developing mouse eye.

Conclusions: : The 32-kD and 46-kD Pax-6 display differential DNA binding activities to the P3 sequence and likely regulate different downstream target genes. Moreover, sumoylation of the 32-kD Pax-6 is necessary to activate its transcriptional activity.

Keywords: cataract • transcription factors • gene/expression