Sumoylation activates the transcriptional activity of Pax-6, an important transcription factor for eye and brain development

Demonstration that sumoylation of the p32 Pax-6 activates its DNA-binding activity. (A) Gel mobility-shifting assay showing that in vitro-translated p32 Pax-6 can bind to the P3 sequence only after preincubation with Pax-6–depleted FHL-124 nuclear extract. (B) Western blot analysis indicating the amounts of in vitro-synthesized mutant and wild-type p32 Pax-6 used for EMSA described in C. (C) EMSA demonstrating that phosphorylation and dephosphorylation modulate DNA binding of p32 Pax-6 to the P3 sequence. (D) EMSA demonstrating that p32 but not p46 Pax-6 binds to the P3 sequence. (E) Western blot analysis of in vitro-synthesized p46 and p32 proteins from Pax-6 full-length cDNA without (lane 1) or with preclearance by serum 11 (lane 2) or by serum 14 (lane 3). (F) EMSA demonstrating that sumoylation is necessary for p32 Pax-6 to bind to the P3 sequence. (G) Demonstration that p32 Pax-6 can be sumoylated by SUMO1 in vitro. (a) The in vitro-generated GST–p32 Pax-6 fusion protein was purified by thrombin cleavage and GST column. (b and c) Purified p32 Pax-6 was subjected to sumoylation with a kit from Biomol (UW8955). After sumoylation, the reaction products were identified by anti-SUMO1 (b) or anti–Pax-6 antibodies (c). Arrowheads indicate p43 Pax-6. (H) EMSA demonstrating that sumoylation of p32 Pax-6 in vitro activates its DNA binding to the P3 sequence.

Demonstration that sumoylation of p32 Pax-6 at K91 activates its DNA-binding and transcriptional activities. (A) EMSA showing that sumoylation at K91 activates p32 Pax-6 DNA-binding activity. (B) EMSA showing that deletion of the N terminus of p32 Pax-6 abolishes its DNA-binding activity (lanes 5 and 10). In contrast, mutation in the other putative sumoylation site, K110, has no effect on the DNA binding of p32 Pax-6 (lane 8). (C) Sumoylation of p32 Pax-6 activates its transactivity on the luciferase reporter gene as tested in FHL-124 cells. (D) Sumoylation of p32 Pax-6 up-regulates expression of the endogenous αB-crystallin gene in ARPE cells. ARPE-19 cells were transfected with vector alone or with wild-type or K91R p32 Pax-6 with or without SUMO1 as indicated. After 36 h, the transfected cells were harvested for preparation of total RNAs, which were used for quantitative real-time PCR as described in Methods. Note that αB-crystallin mRNA from vector-transfected cells is considered as 1.0. Transfection of wild-type p32 Pax-6 yielded a 12-fold increase in αB-crystallin mRNA expression. Cotransfection of the wild-type p32 Pax-6 with SUMO1 leads to an additional 11-fold enhancement of the αB-crystallin mRNA expression. The K91R mutant substantially decreased its transactivity. (E) ChIP assay demonstrating that Pax-6 sumoylated by SUMO1 binds directly to the αB-crystallin gene promoter in ARPE-19 cells. Lane 5 indicates ChIP result from sequential precipitations, first by anti-SUMO1 and then by anti–Pax-6 antibodies. (F) Sumoylation of p32 Pax-6 up-regulates expression of the endogenous Cspg2 and Mab2112 genes in αTN4-1 cells. αTN4-1 cells were transfected and processed as described in Fig. 3D. Cotransfection of the wild-type p32 Pax-6 with SUMO1 leads to a 2.2-fold enhancement of Cspg2 mRNA expression and a 5.1-fold increase of Mab2112 mRNA expression in αTN4-1 cells. The p32 K91R mutant substantially decreased its transactivity.