Fig 2: COP1 interacts with Sox2 directly. a Cells were transfected with indicated constructs and siRNA. Flag-Sox2 was immunoprecipated with anti-Flag and immunoblotted with anti-Myc. b HEK293T cells were transfected with indicated constructs and siRNA, and Sox2 was immunoprecipated with anti-Flag and immunoblotted with anti-Myc. c HEK293T cells were transfected with indicated constructs and western blot was performed to measure the expression of Flag-Sox2, Flag-COP1, and Myc-DET1. d Increasing amounts of Flag-COP1 were co-transfected together with Flag-Sox2 and DET1 into HEK293T cells and ectopic Flag-Sox2 expression was detected. e The predicted work model of CUL4ADET1-COP1 for Sox2 degradation. The representative images are shown from three independent experiments. Unprocessed original scans of blots are shown in Supplementary Fig. 9

Fig 3: CUL4ADET1-COP1 ubiquitylates SOX2. a HEK293T cell line with knockdown of COP1 (upper panel) or DET1 (lower panel) were treated with CHX (10 µg/ml), and collected at the indicated times for western blot. Quantification of Sox2 level relative to tubulin is shown. Results are shown as mean ± s.d. n = 3 independent experiments. **P < 0.01, two-way ANOVA test. b HA-Ub was co-transfected with indicated siRNA into HEK293T cells. Cells were treated with MG132 for 8 h before collection. Sox2 was immunoprecipitated with anti-Sox2 and the ubiquitylated Sox2 proteins were immunoblotted with anti-HA. c Cell-free Sox2 ubiquitylation assay. The purified GST-Sox2, CUL4A, COP1, and DET1 proteins were incubated with commercial E1, UbE2D3 (E2), and ubiquitin for 2 h at 37 °C. The mixtures were subjected to GST pull-down and western blot with anti-His antibody. d Immunoblotting of Flag-Sox2 in cells transfected with indicated constructs. Quantification of Sox2 level is shown under the blot. e Cells were transfected with indicated siRNA and the cell lysates were subjected to western blot to detect Sox2 expression. Quantification of relative protein level is shown under the blot. The representative images are shown from three independent experiments. Unprocessed original scans of blots are shown in Supplementary Fig. 9

Fig 4: COP1 and OTUD7B coordinate to control NPCs differentiation. a, b NPCs were plated in Matrigel-coated six-well plate for neuronal differentiation. For the indicated times, immunoblotting (a) and immunofluorescence (b) of Sox2, CUL4A, COP1, OTUD7B and TUJ1 were performed. c Cells transfected with HA-Ub and indicated shRNA were treated by MG132 for 8 h, and the lysates were immunoprecipated with anti-Sox2 antibody and immunoblotted with anti-HA. d Summary of our findings. OTUD7B stabilizes Sox2 through deubiquitylation and maintains the stemness of NPCs, while CUL4ADET1-COP1 promotes Sox2 degradation through ubiquitylation and induces differentiation of NPCs. OTUD7B and CUL4ADET1-COP1 exert opposite roles in regulating Sox2 protein stability at the post-translational level. The representative images are shown from three independent experiments. Unprocessed original scans of blots are shown in Supplementary Fig. 9

Fig 5: COP1 promotes NPCs differentiation in a Sox2-dependent manner. a NPCs were plated in Matrigel-coated six-well plate for neuronal differentiation. For the indicated times, fixed and stained with anti-Sox2 (green), anti-COP1 (red), anti-TUJ1 (red), or CUL4A (green)-specific antibodies. Nuclei were counterstained with Hoechst 33342 (HO, blue). Scale bar, 50 µm. b, c NPCs were infected with lentiviruses expressing COP1 shRNA or negative control (NC) shRNA for 126 h. Immunoblotting (b) and immunostaining (c) were performed for Sox2, COP1, TUJ1, or Nestin. Scale bar, 50 µm. d NPCs with double knockdown of COP1 and Sox2 were generated by lentivirus infection. Immunostaining were performed for Sox2, TUJ1. Scale bar, 50 µm. The ratio of TUJ1-positive or Sox2-positive cells were quantified. Results are shown as mean ± s.d. Each error bar shows the standard deviation of numbers of positive cells in 10 fields of view. *P < 0.05, Student’s t-test. e NPCs with co-expression of COP1 and Sox2-AA were generated by lentivirus infection. Cas9-based activator of COP1 (SAM-COP1) were used to activate COP1 expression in NPCs. Immunostaining were performed for Sox2 and TUJ1. Scale bar, 50 µm. The ratio of TUJ1-positive or Sox2-positive cells were quantified. Results are shown as mean ± s.d. Each error bar shows the standard deviation of numbers of positive cells in 10 fields of view. *P < 0.05, Student’s t-test. The representative images are shown from three independent experiments. Unprocessed original scans of blots are shown in Supplementary Fig. 9