Fig 2: Tumor suppressor genes E-cadherin and FBXW7 are cotargeted by PHF1/PRMT5/CRL4B complex. (A) The efficiency of four shRNAs targeting PHF1, PRMT5 or CUL4B, respectively. MDA-MB-231 cells were infected with lentiviruses carrying control shRNA (shSCR) or four different shRNAs targeting PHF1, PRMT5 or CUL4B. The knockdown efficiencies of PHF1, PRMT5, and CUL4B were verified by RT-qPCR. We chose shPHF1#2, shPRMT5#3, and shCUL4B#2 (marked in red) for further study. Error bars represent mean ± SD for three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed unpaired t test). (B and C) Clones in which PHF1, PRMT5, or CUL4B were stably knocked down were compared with the parental cell lines with respect to the levels of mRNA and protein of E-cadherin and FBXW7 in MDA-MB-231, Hs 578T and MDA-MB-453 cells. The mRNA levels were normalized to those of GAPDH and β-actin served as a loading control for western blotting. Error bars represent mean ± SD of three independent experiments. (∗P < 0.05, ∗∗P < 0.01, and two-tailed unpaired t test). (D) PHF1, PRMT5–WDR77, and CRL4B complexes exist in the same protein complex on the E-cadherin and FBXW7 promoters. CUL4A served as a negative control. ChIP and Re-ChIP experiments were performed in MDA-MB-231 cells with the indicated antibodies. (E) qChIP analysis of the recruitment of the indicated proteins on E-cadherin and FBXW7 promoters in MDA-MB-231 cells after transfection with control shRNA or shRNAs targeting PHF1, PRMT5 or CUL4B. Purified rabbit IgG was used as a negative control. Error bars represent mean ± SD of three independent experiments (∗P < 0.05, ∗∗P < 0.01, and two-tailed unpaired t test).

Fig 3: CUL4ADET1-COP1 interacts with Sox2 and regulates its stability. a Network view of E3–Sox2 interactions (left panel) and the E3 hierarchical tree for Sox2 (right panel). UbiBrowser was employed to explore the E3 ligases for Sox2. The representative predicted E3 ligases surround Sox2. The node colors and characters reflect the E3 type. The edge width, the node size, and the edge shade are corrected with the confidence score. The predicted E3s and their position in the E3 family hierarchical tree was presented. In this tree, texts in each circle (just like “U”, “D” and “SO”) represent the E3 family. The number in the bracket following each E3 family represents the number of corresponding predicted E3–Sox2 interaction. b NPCs cell lysates were subjected to immunoprecipitation with control IgG or anti-Sox2 antibodies and detected CUL4A, COP1, DET1, DDB1, Roc1, and Sox2 protein levels. c The lysates of HEK293T cells transfected with indicated constructs were subjected to immunoprecipitation with anti-Myc or Histidine tag-specific affinity resin (agarose beads). The immunoprecipitates or the eluates were then blotted. d Overview of the structures of COP1 wild type and different truncates. HEK293T cells were co-transfected with Myc-Sox2 and the indicated COP1 truncates. The lysates were collected and subjected to immunoprecipitation with anti-Flag. The immunoprecipitates were then blotted. e Overview of the structure of Sox2 wild type and different VP mutants. Recombinant proteins (His-COP1, GST-Sox2, GST-Sox2-A1, GST-Sox2-A2, and GST-Sox2-AA) were expressed and purified. GST-Sox2 bound to glutathione-Sepharose 4B beads was incubated with His- COP1 for 24 h at 4 °C. Then the beads were washed and proteins were eluted, followed by western blotting. f HEK293T cells were transfected with indicated constructs. The lysates were collected and blotted with anti-Flag and anti-Myc antibody. The representative images are shown from three independent experiments. Unprocessed original scans of blots are shown in Supplementary Fig. 9

Fig 4: CUL4A contributes to Sox2 ubiquitylation in NPCs differentiation. a NPCs (neural stem/progenitor cells derived from human NPSCs, WA09) were cultured in neural induction medium (NIM) to undergo cellular differentiation for the indicated times. NPCs were treated with 10 µg per ml CHX, and collected at the indicated times for western blot. Quantification of Sox2 levels relative to tubulin is shown. Results are shown as mean ± s.d. n = 3 independent experiments. **P < 0.01, two-way ANOVA test. b NPCs transfected with HA-Ub were treated with MG132 for 8 h before collection. Sox2 was immunoprecipitated with anti-Sox2 antibody and immunoblotted with anti-HA antibody. c Immunoblotting of Sox2, CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5, and TUJ1 during NPCs differentiation. d Immunoblotting of Sox2 in HEK293T cells transfected with indicated siRNA. NC represents the empty vector control. e HEK293T cells were transfected with indicated constructs and the level of Flag-Sox2 was detected by immunoblotting. NC represents the empty vector control. f, g HA-Ub was co-transfected together with indicated siRNA (f) or constructs (g) into HEK293T cells. Cells were treated with MG132 for 8 h before collection. Then Sox2 was immunoprecipitated with anti-Sox2 antibody and immunoblotted with anti-HA antibody. NC represents the empty vector control. h Immunoblotting of Sox2, KLF4 and E2F1 in MEFs with CUL4A, CUL4B knockout or WT control. Quantification of protein levels relative to vinculin is shown. i MEFs were treated with CHX (10 µg/ml), and collected at the indicated times for western blot. Quantification of Sox2 levels relative to vinculin is shown. **P < 0.01, two-way ANOVA test. 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