Fig. 1Sumoylation of Hes6. (A) HeLa cells were co-transfected with Hes6 and RFP-SUMO paralogues (SUMO1 or SUMO3) in the presence or absence of Ubc9. Samples were analyzed by Western blot using anti-Hes6 antibodies. (B) Hes6, RFP-SUMO, and Ubc9 were co-expressed in cells and the cell extracts were subjected to immunoprecipitation followed by Western blot using anti-Hes6, anti-SUMO1, or anti-SUMO3 antibodies. Arrowheads indicate the sumoylated Hes6 (black) or native Hes6 (white) band.
Fig. 2Hes6 is sumoylated at lysine residues 27 and 30. (A) Proteins were extracted from HeLa cells transfected with Hes6 constructs encoding wild type (WT) or its Lys-Arg mutants. Western blot was performed with the indicated antibodies. (B) Cells were transfected with Hes6 constructs encoding WT, the single mutants (K27R or K30R), or the 2KR (K27/30R) double mutant together with SUMO and Ubc9 and analyzed by Western blot using anti-Hes6 antibodies.
Fig. 3Sumoylation promotes ubiquitination of Hes6. (A) HeLa cells expressing wild type (WT) Hes6 or the 2KR (K27/30R) double mutant were treated with cychloheximide (CHX, 30 µg/mL) for indicated time periods. The protein abundance of Hes6 was analyzed by Western blot using anti-Hes6 antibodies and then quantitated by densitometer. (B) Cells were co-transfected with WT Hes6 or the 2KR mutant and HA-tagged ubiquitin (Ub) in the presence or absence of RFP-SUMO and SUSP1 and incubated for 5 hours with 25 µM MG132. Cell extracts were analyzed by immunoprecipitation followed by Western blot using anti-HA, anti-SUMO, or anti-Hes6 antibodies.
Fig. 4Sumoylation regulates rhythmic expression of Hes6. (A) The schematic diagram of GFP-fused wild type (WT) or the 2KR (K27/30R) mutant Hes6 under the control of the Hes6 promoter. (B) NIH 3T3 cells were transfected with GFP-Hes6 constructs encoding WT or the 2KR mutant. After serum treatment (t=0), Hes6 protein levels were examined every 30 minutes.
Fig. 5Effects of Hes6 sumoylation on Hes1-induced transcriptional suppression. (A) HeLa cells were co-transfected with pHes1-luciferase reporter, Hes1 (20 ng), and increasing amounts of Hes6 (50, 100, 200, and 300 ng). After overnight incubation, the bioluminescence was measured using a luminometer. Each value is the mean±SEM of three independent experiments. (B) Cells were co-transfected with the pHes1-luciferase reporter, Hes1, and Hes6 (wild type [WT] or the 2KR [K27/30R] mutant). Each value is the mean±SEM of three independent experiments (P<0.001). (C) Cells were co-transfected with Hes1 and Hes6 and the cell lysates were immunoprecipitated using anti-Hes1 antibodies. (D) Chromatin was extracted from HeLa cells transfected with Hes1 and Hes6 constructs encoding WT or the 2KR mutant. Chromatin immunoprecipitation (ChIP) assays were performed with the indicated antibodies. aP<0.001 vs. Hes1 expression; bP<0.001.